vvEPA
United States
Environmental Protection
Agency
Method B: Bacteroidales in Water by
TaqMan® Quantitative Polymerase Chain
Reaction (qPCR) Assay
June 2010
Note that this method will be updated following its validation in marine
and fresh ambient waters.
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-822-R-10-003
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Acknowledgments
This method was developed under the direction of Rich Haugland, Kevin Oshima and Alfred P. Dufour of
the U.S. Environmental Protection Agency's (EPA) Human Exposure Research Division, National
Exposure Research Laboratory, Cincinnati, Ohio. Screen shots for the ABI 7500 and the Smart Cycler®
(Software version 2.0) were kindly provided by Jack Paar III of EPA's New England Regional
Laboratory.
The following laboratories are gratefully acknowledged for their participation in the single laboratory
validation of this method in fresh and marine waters:
Participant Laboratories
• New York State Department of Health, Environmental Biology Laboratory: Ellen Braun-Howland
and Stacey Chmura
• Mycometrics, LLC: King-Teh Lin and Pi-shiang Lai
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Disclaimer
Neither the United States Government nor any of its employees, contractors, or their employees make any
warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use of
apparatus, product, or process discussed in this method, or represents that its use by such party would not
infringe on privately owned rights. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Questions concerning this method or its application should be addressed to:
Robin K. Oshiro
Engineering and Analysis Division (4303T)
U.S. EPA Office of Water, Office of Science and Technology
1200 Pennsylvania Avenue, NW
Washington, DC 20460
oshiro.robin@epa.gov or OSTCWAMethods@epa.gov
IV
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Introduction
Bacteria of the Bacteroidales order are commonly found in the feces of humans and other warm-blooded
animals. Although these organisms can be persistent in the environment, the presence of Bacteroidales in
water is an indication of fecal pollution and the possible presence of enteric pathogens.
Method B describes a quantitative polymerase chain reaction (qPCR) procedure for the detection of DNA
from Bacteroidales bacteria in ambient water matrices based on the amplification and detection of-a
specific region of the 16S ribosomal RNA gene from these organisms. Results can be obtained by this
method in 3-4 hours, allowing same-day notification of recreational water quality. Recent epidemiological
studies at fresh water recreational beaches (Reference 17.7) have demonstrated similar or improved
positive correlations between Bacteroidales DNA measurements by this method and swimming-
associated gastrointestinal (GI) illness rates.
In Method B, water samples are filtered to collect Bacteroidales on polycarbonate membrane filters.
Following filtration, total DNA is solubilized from the filter retentate using a bead beater. Bacteroidales
target DNA sequences present in the clarified homogenate are detected by the real time polymerase chain
reaction technique using TaqMan® Universal Master Mix PCR reagent and the TaqMan® probe system.
The TaqMan® system signals the formation of PCR products by a process involving enzymatic
hydrolysis of a fluorogenically-labeled oligonucleotide probe when it hybridizes to the target sequence.
Method B uses an arithmetic formula, the comparative cycle threshold (CT) method, to calculate the ratio
of Bacteroidales 16S rRNA gene target sequences (target sequences) recovered in total DNA extracts
from water samples relative to those in similarly-prepared extracts of calibrator samples containing a
known quantity of Bacteroidales cells. Mean estimates of the absolute quantities of target sequences in
the calibrator sample extracts are then used to determine the absolute quantities of target sequences in the
water samples. CT values for sample processing control (SPC) sequences added in equal quantities to
both the water filtrate and calibrator samples before DNA extraction are used to normalize results for
potential differences in DNA recovery or to signal inhibition or fluorescence quenching of the PCR
analysis caused by a sample matrix component or possible technical error.
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Method 1
3.0 Definitions 1
4.0 Interferences 2
5.0 Safety 3
6.0 Equipment and Supplies 3
7.0 Reagents and Standards 5
8.0 Sample Collection, Handling, and Storage 9
9.0 Quality Control 9
10.0 Calibration and Standardization of Method-Related Instruments 12
11.0 Procedure 12
12.0 Data Analysis and Calculations 21
13.0 Sample Spiking Procedure 24
14.0 Method Performance 25
15.0 Pollution Prevention 25
16.0 Waste Management 25
17.0 References 26
18.0 Acronyms 26
VI
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List of Appendices
Appendix A: ABI 7900 and ABI 7500 Sequence Detector Operation
Appendix B: Cepheid Smart Cycler® Operation
vi i
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Method B: Bacteroidales in Water by Quantitative Polymerase Chain
Reaction (qPCR) Assay
June 2010
1.0 Scope and Application
1.1 Method B describes a quantitative polymerase chain reaction (qPCR) procedure for the
measurement of the 16S ribosomal RNA (16S rRNA) target gene sequences from all known
bacteria of the order Bacteroidales in water. This method is based on the collection of
Bacteroidales on membrane filters, extraction of total DNA using a bead beater, and detection of
Bacteroidales target sequences in the supernatant by real time polymerase chain reaction using
TaqMan® Universal Master Mix PCR reagent and the TaqMan® probe system. The TaqMan®
system signals the formation of PCR products by a process involving the enzymatic hydrolysis of
a labeled fluorogenic probe that hybridizes to the target sequence.
1.2 Bacteroidales are commonly found in the feces of humans and other warm-blooded animals.
Although DNA from these organisms can be persistent in the environment, its presence in water
is an indication of fecal pollution and the possible presence of enteric pathogens.
1.3 The Method B test is recommended as a measure of ambient fresh and marine recreational water
quality. Epidemiological studies have been conducted at fresh water and marine water beaches
that may lead to the potential development of criteria that can be used to promulgate recreational
water standards based on established relationships between health effects and water quality
measurements by this method. The significance of finding Bacteroidales DNA target sequences
in recreational water samples stems from the direct relationship between the density of these
sequences and the risk of gastrointestinal illness associated with swimming in the water that have
been observed thus far in the epidemiological studies (Reference 17.7).
1.4 This method assumes the use of an ABI sequence detector as the default platform. The Cepheid
Smart Cycler® may also be used. The user should refer to the platform specific instructions for
these instruments in the Appendices. Users should thoroughly read the method in its entirety
before preparation of reagents and commencement of the method to identify differences in
protocols for different platforms.
2.0 Summary of Method
The method is initiated by filtering a water sample through a membrane filter. Following
filtration, the membrane containing the bacterial cells and DNA is placed in a microcentrifuge
tube with glass beads and buffer, and then agitated to extract the DNA into solution. The
clarified supernatant is used for PCR amplification and detection of target sequences using the
TaqMan® Universal Master Mix PCR reagent and probe system.
3.0 Definitions
3.1 Bacteroidales: all genera and species of the order Bacteroidales for which 16S rRNA gene
nucleotide sequences were reported in the GenBank database
(http://www.ncbi.nlm.nih/gov/Genbank) at the time of method development.
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Method B
3.2 Target sequence: A segment of the 16S rRNA gene containing nucleotide sequences that are
homologous to both the primers and probe used in the Bacteroidales qPCR assay and that are
common only to species within this order.
3.3 Sample processing control (SPC) sequence (may also be referred to as reference sequence): A
segment of the ribosomal RNA gene operon, internal transcribed spacer region 2 of chum salmon,
Oncorhynchus keta (O. keta), containing nucleotide sequences that are homologous to the primers
and probe used in the SPC qPCR assay. SPC sequences are added as part of a total salmon DNA
solution in equal quantities to all water sample filtrate and calibrator samples prior to extracting
DNA from the samples.
3.4 DNA standard: A purified, RNA-free and spectrophotometrically quantified and characterized
Bacteroides thetaiotaomicron strain ATCC #29741 genomic DNA preparation. DNA standards
are used to generate standard curves for determination of performance characteristics of the qPCR
assays and instrument with different preparations of master mixes containing TaqMan® reagent,
primers and probe as described in Section 9.6. Also used for quantifying target sequences in
calibrator sample extracts as described in Section 12.2.
3.5 Calibrator sample: Samples containing constant added quantities of B. thetaiotaomicron strain
ATCC #29741 cells and SPC sequences that are extracted and analyzed in the same manner as
water samples. Calibrator sample analysis results are used as positive controls for the
Bacteroidales target sequence and SPC qPCR assays and as the basis for target sequence
quantification in water samples using the AACT or ACT comparative cycle threshold calculation
method as described in Section 12.4. Analysis results of these samples provide corrections for
potential daily or weekly method-related variations in Bacteroidales cell lysis, target sequence
recovery and PCR efficiency. QPCR analyses for SPC sequences from these samples are also
used to correct for variations in total DNA recovery in the extracts of water sample filtrates that
can be caused by contaminants in these filtrates as described in Section 12.4 and/or to signal
potentially significant PCR inhibition caused by these contaminants as described in Section 9.8..
3.6 AACT comparative cycle threshold calculation method (AACT method): A calculation method
derived by Applied Biosy stems (Reference 17.1) for calculating the ratios of target sequences in
two DNA samples (e.g., a calibrator and water sample) that either controls (ACT method) or
normalizes (AACT method) for differences in total DNA recovery from these samples using qPCR
analysis CT values for a reference (SPC) sequence that is initially present in equal quantities prior
to DNA extraction.
3.7 Amplification factor (AF): A measure of the average efficiency at which target or SPC sequences
are copied and detected by their respective primer and probe assays during each thermal cycle of
the qPCR reaction that is used in the comparative cycle threshold calculation methods. AF values
can range from 1 (0 % of sequences copied and detected) to 2 (100 % of sequences copied and
detected) and are calculated from a standard curve as described in Section 12.2.
4.0 Interferences
Water samples containing colloidal or suspended particulate materials can clog the membrane
filter and prevent filtration. These materials can also interfere with DNA recovery and interfere
with the PCR analysis by inhibiting the enzymatic activity of the Taq DNA polymerase, and/or
inhibiting the annealing of the primer and probe oligonucleotides to sample target DNA enzyme
or quenching of hydrolyzed probe fluorescence.
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Method B
5.0 Safety
5.1 The analyst/technician must know and observe the normal safety procedures required in a
microbiology and/or molecular biology laboratory while preparing, using, and disposing of
cultures, reagents, and materials, and while operating sterilization equipment.
5.2 Where possible, facial masks should be worn to prevent sample contamination.
5.3 Mouth-pipetting is prohibited.
6.0 Equipment and Supplies
6.1 Separated, and dedicated workstations for reagent preparation and for sample preparation,
preferably with HEPA-filtered laminar flow hoods and an Ultraviolet (UV) light source, each
having separate supplies (e.g., pipettors, tips, gloves, etc.). Note: The same workstation may be
used for the entire procedure provided that it has been cleaned with bleach and UV sterilized as
specified in section 11.6.1 between reagent and sample preparation. Under ideal conditions, two
dedicated workstations are used for sample preparation: one for preparing samples with high
target sequence DNA concentrations (e.g., DNA standards and calibrator samples) and one for
preparing samples with expected low target sequence DNA concentrations (e.g., filter blanks and
ambient water samples).
6.2 Balance capable of accuracy to 0.01 g
6.3 Extraction tubes: semi-conical, screw cap microcentrifuge tubes, 2.0-mL (e.g., PGC 506-636 or
equivalent)
6.4 Glass beads, acid washed, 212 - 300 um (e.g., Sigma G-1277 or equivalent)
6.5 Autoclave, capable of achieving and maintaining 121°C [15 Ib pressure per square inch (PSI)] for
minimally 15 minutes
6.6 Workstation for water filtrations, preferably a HEPA-filtered laminar flow hood with a UV light
source. This can be the same as used for sample preparation, Section 6.1
6.7 Sterile bottles/containers for sample collection
6.8 Membrane filtration units (filter base and funnel) for 47 mm diameter filters, sterile glass, plastic
(e.g. Pall Gelman 4242, or equivalent), stainless steel, or disposable plastic (e.g., Nalgene CN
130-4045 or CN 145-0045, or equivalent), cleaned and bleach treated (rinsed with 10% v/v
bleach, then 3 rinses with reagent-grade water), covered with aluminum foil or Kraft paper, and
autoclaved or UV-sterilized if non-disposable
6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source. In an emergency or
in the field, a hand pump or a syringe equipped with a check valve to prevent the return flow of
air can be used.
6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing
6.11 Filter manifold to hold a number of filter bases
6.12 Flask for safety trap placed between the filter flask and the vacuum source
6.13 Anaerobic chamber (BD BBL GasPak 150 Jar System or equivalent)
6.14 Disposable gas generator pouches (BD BBL Gas Pak Plus Hydrogen or equivalent)
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Method B
6.15 Forceps, straight or curved, with smooth tips to handle filters without damage, 2 pairs
6.16 Polycarbonate membrane filters, sterile, white, 47 mm diameter, with 0.45 /u,m pore size
(e.g., GE Osmotics Inc. 04CP04700 or equivalent)
6.17 Graduated cylinders, 100-1000-mL, cleaned and bleach treated (rinsed with 10% v/v bleach, then
3 rinses with reagent-grade water), covered with aluminum foil or Kraft paper and autoclaved or
UV-sterilized
6.18 Petri dishes, sterile, plastic or glass, 100 x 15 mm with loose fitting lids
6.19 Disposable loops, 1 and 10 uL
6.20 Sterile Ice syringes
6.21 Sterile Va" needles, 18 gauge
6.22 Permanent ink marking pen for labeling tubes
6.23 Visible wavelength spectrophotometer capable of measuring at 595 nm
6.24 Single or 8-place mini bead beater (e.g., Biospec Products Inc. #3110BX or equivalent).
6.25 Microcentrifuge capable of 12,000 x g
6.26 Micropipettors with 10, 20, 200 and 1000 uL capacity. Under ideal conditions, each workstation
should have a dedicated set of micropipettors (one micropipettor set for pipetting reagents not
containing cells or reference DNA and one set for reagents containing reference DNA and for test
samples).
6.27 Micropipettor tips with aerosol barrier for 10, 20, 200 and 1000 uL capacity micropipettors.
Note: All micropipetting should be done with aerosol barrier tips. The tips used for reagents not
containing DNA should be separate from those used for reagents containing DNA and test
samples. Each workstation should have a dedicated supply of tips.
6.28 Microcentrifuge tubes, low retention, clear, 1.7-mL (e.g., GENE MATE C-3228-1 or equivalent)
6.29 Test tube rack for microcentrifuge tubes, use a separate rack for each set of tubes
6.30 Conical centrifuge tubes, sterile, screw cap, 50-mL
6.31 Test tubes, screw cap, borosilicate glass, 16 x 125 mm
6.32 Pipet containers, stainless steel, aluminum or borosilicate glass, for glass pipets
6.33 Pipets, sterile, T.D. bacteriological or Mohr, disposable glass or plastic, of appropriate volume
(disposable pipets preferable)
6.34 Vortex mixer (ideally one for each work station)
6.35 Dedicated lab coats for each work station
6.36 Disposable powder-free gloves for each work station
6.37 Refrigerator, 4°C (ideally one for reagents and one for DNA samples)
6.38 Freezer, -20°C or -80°C (ideally one for reagents and one for DNA samples)
6.39 Ice, crushed or cubes for temporary preservation of samples and reagents
6.40 Printer (optional)
6.41 Data archiving system (e.g., flash drive or other data storage system)
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Method B
6.42 UV spectrophotometer capable of measuring wavelengths of 260 and 280 nm using small volume
capacity (e.g., 0.1 mL) cuvettes or NanoDrop® (ND-1000) spectrophotometer (or equivalent)
capable of the same measurements using 2 uL sample volumes
6.43 ABI 7900 or ABI 7500 Sequence Detector
6.43.1 Optical 96 well PCR reaction tray (e.g., Applied Biosystems N801-0560 or equivalent)
6.43.2 Optical adhesive PCR reaction tray tape (e.g., Applied Biosystems 4311971 or
equivalent) or MicroAmp™ caps (e.g., Applied Biosystems N8010534 or equivalent)
6.43.3 ABI 7900 sequence detector
6.44 Cepheid Smart Cycler®
6.44.1 Smart Cycler® 25 uL PCR reaction tubes (e.g., Cepheid 900-0085 or equivalent)
6.44.2 Rack and microcentrifuge for Smart Cycler® PCR reaction tubes. Note: Racks and
microcentrifuge are provided with the Smart Cycler® thermocycler
6.44.3 Cepheid Smart Cycler® System Thermocycler
7.0 Reagents and Standards
Note: The Bacteroidales stock culture (Section 7.9), Salmon DNA/extraction buffer (Section
7.13), and DNA extraction tubes (Section 7.19), may be prepared in advance.
7.1 Purity of Reagents: Molecular-grade reagents and chemicals shall be used in all tests
7.2 Control Culture
• Bacteroides thetaiotaomicron (B. thetaiotaomicron) ATCC #29741
7.3 Sample Processing Control (SPC) DNA (source of SPC control sequences)
• Salmon testes DNA (e.g., Sigma D1626 or equivalent)
7.4 Phosphate Buffered Saline (PBS)
7.4.1 Composition:
Monosodium phosphate (NaH2PO4) 0.58 g
Disodium phosphate (Na2HPO4) 2.50 g
Sodium chloride 8.50 g
Reagent-grade water 1.0 L
7.4.2 Dissolve reagents in 1 L of reagent-grade water in a flask and dispense in appropriate
amounts for dilutions in screw cap bottles or culture tubes, and/or into containers for use
as reference matrix samples and rinse water. Autoclave after preparation at 121°C [15 Ib
pressure per square inch (PSI)] for 15 minutes. Final pH should be 7.4 ± 0.2.
7.5 Chopped Meat Carboyhydrates Broth (CMCB) Note: Formulation is provided only to ensure that
the appropriate medium is used for analyses, laboratories should use commercially prepared
tubes.
Note: Pre-mixed powder forms of this medium are not available
7.5.1 Composition:
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Method B
Chopped Meat Pellets 10.2 g
Pancreatic Digest of Casein 30.0 g
Yeast extract 5.0g
Glucose 4.0 g
Dipotassium Phosphate 5.0g
Cellobiose l.Og
Maltose l.Og
Starch l.Og
L-Cysteine HC1 0.5 g
Resazurin 0.001 g
Vitamin K] 1.0 mg
Hemin 5.0 mg
Reagent-grade water l.OL
7.5.2 This medium is manufactured in a pre-reduced (oxygen-free) environment and sealed to
prevent aerobiosis. The medium is pre-sterilized and ready for inoculation.
7.6 CDC Anaerobe 5% Sheep Blood (BAP) Note: Formulation is provided only to ensure that the
appropriate medium is used for analyses, laboratories should use commercially prepared plates.
7.6.1 Composition:
Pancreatic Digest of Casein 15.0 g
Papaic Digest of Soybean Meal 5.0 g
Sodium Chloride 5.0 g
Agar 20.0 g
Yeast Extract 5.0g
Hemin 0.005 g
Vitamin K! 0.01 g
L-Cystine 0.4 g
Reagent-grade water 1 L
Defibrinated Sheep Blood 5%
7.7 Sterile glycerol (used for preparation of B. thetaiotaomicron stock culture as described in section
7.9)
7.8 Preparation of B. thetaiotaomicron (ATCC #29741) stock culture
Rehydrate lyophilized B. thetaiotaomicron per manufacturer's instructions (for ATCC stocks,
suspend in 0.5 mL of sterile chopped meat carbohydrate broth (CMCB) and mix well to dissolve
lyophilized culture. Using a sterile syringe and needle aspirate suspension and inoculate 10 mL of
CMCB. Incubate at 35.0°C ± 0.5°C for 24-72 hours. After incubation remove septum and
transfer liquid to a sterile tube by pipetting. While pipetting the suspension use extreme care to
remove as much liquid as possible without siphoning any particulates. Centrifuge suspension at
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Method B
6000 x g for 5 minutes to create a cell pellet. Using a sterile pipet, discard supernatant.
Resuspend pellet in 10 mL of fresh sterile CMCB without the particulates containing 15%
glycerol and dispense in 1.5-mL aliquots in microcentrifuge tubes. Freeze at -20°C (short term
storage) or -80°C (long term storage). Note: Aliquots of suspension may be plated to determine
CPU concentration as described in Section 11.1. It is advisable to verify the B. thetaiotaomicron
culture by using commercially available test kits (e.g., Vitek® or API®)
7.9 PCR-grade water (e.g., OmniPurwater from VWREM-9610 or equivalent). Watermustbe
DNA/DNase free.
7.10 Isopropanol or ethanol, 95%, for flame-sterilization
7.11 AE Buffer, pH 9.0 (e.g., Qiagen 19077 or equivalent) (Note: pH 8.0 is acceptable)
Composition:
lOmMTris-Cl (chloride)
0.5 mM EDTA (Ethylenediaminetetraacetic acid)
7.12 Salmon DNA/extraction buffer
7.12.1 Composition:
Stock Salmon testes DNA (10 ug/mL) (Section 7.3)
AE Buffer (Section 7.12)
7.12.2 Preparation of stock Salmon testes DNA: Each bottle of Salmon DNA contains a specific
number of units. Note the units. Add an equal volume of PCR-grade water to dissolve
the Salmon testes DNA, and stir, using a magnetic stir bar at low to medium speed, until
dissolved (2-4 hours if necessary). The solution at this point will be equivalent to 50 ug
Salmon testes DNA/mL. Dilute using PCR grade water to a concentration of ~ 10
ug/mL. Determine concentration of Salmon testes DNA stock by OD26o reading in a
spectrophotometer. An OD26o of 1 is approximately equal to 50 ug/mL (one Unit). This
is your Salmon testes DNA stock solution. Unused portion may be aliquoted and frozen
at-20°C.
Note: For example, if the bottle contains 250 mg of DNA, using sterilized scissors and
sterilized forceps, cut a piece of DNA to weigh approximately 20 mg (approximately 304
Units), and place in a sterile weigh boat. After weighing, place the DNA into a sterile 50
mL tube and add 20 mL PCR grade water. Cap tightly, and resuspend by 2-4 hours of
gentle rocking. The concentration should be about 1 mg/mL. Remove three, 10 uL
aliquots and dilute each to 1 mL with PCR grade water. Check absorbance (OD26o) and
calculate DNA using the assumption 1 Unit DNA is equal to 1 OD26o, which is then
equivalent to 50 ug/mL DNA. Adjust this stock to 10 ug/mL based on calculated initial
concentration of 1 mg/mL by diluting with PCR grade water. Aliquot portions of the
adjusted DNA stock and freeze.
7.12.3 Dilute Salmon testes DNA stock with AE buffer to make 0.2 ug/mL Salmon
DNA/extraction buffer. Extraction buffer may be prepared in advance and stored at 4° C
for a maximum of 1 week.
Note: Determine the total volume of Salmon DNA/extraction buffer required for each day
or week by multiplying volume (600 uL) * total number of samples to be analyzed
including controls, water samples, and calibrator samples. For example, for 18 samples,
prepare enough Salmon/DNA extraction buffer for 24 extraction tubes (18^-6 = 3,
therefore, 3 extra tubes for water sample filtration blanks (method blanks) and 3 extra
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Method B
tubes for calibrator samples). Note that the number of samples is divided by 6 because
you should conduct one method blank for every 6 samples analyzed. Additionally,
prepare excess volume to allow for accurate dispensing of 600 uL per tube, generally 1
extra tube. Thus, in this example, prepare sufficient Salmon/DNA extraction buffer for
24 tubes plus one extra. The total volume needed is 600 uL x 25 tubes = 15,000 uL.
Dilute the Salmon testes DNA working stock 1:50, for atotal volume needed (15,000 uL)
+ 50 = 300 uL of 10 ug/mL Salmon testes DNA working stock. The AE buffer needed is
the difference between the total volume and the Salmon testes DNA working stock. For
this example, 15,000 uL - 300 uL = 14,700 uL AE buffer needed.
7.13 Bleach solution: 10% v/v bleach (or other reagent that hydrolyzes DNA) (used for cleaning work
surfaces)
7.14 Sterile water (used as rinse water for work surface after bleaching)
7.15 TaqMan® Universal PCR Master Mix (e.g., Applied Biosystems 4304437 or equivalent)
Composition:
AmpliTaq Gold® DNA Polymerase
AmpErase® UNO
dNTPs with dUTP
Passive Reference 1 (ROX™ fluorescent dye)
Optimized buffer components (KC1, Tris, EDTA, MgCl2)
7.16 Bovine serum albumin (BSA), fraction V powder e.g., Sigma B-4287 or equivalent)
Dissolve in PCR-grade water to a concentration of 2 mg/mL.
7.17 Primer and probe sets: Primer and probe sets may be purchased from commercial sources.
Primers should be desalted, probes should be HPLC purified.
7.17.1 Bacteroidales primer and probe set (References 17.4 and 17.6):
Forward primer: 5'-GGGGTTCTGAGAGGAAGGT
Reverse primer: 5'-CCGTCATCCTTCACGCTACT
TaqMan® probe: 5'-FAM-CAATATTCCTCACTGCTGCCTCCCGTA-TAMRA
7.17.2 Salmon DNA primer and probe set (Reference 17.4):
Forward primer: 5'-GGTTTCCGCAGCTGGG
Reverse primer (Sketa 22): 5'-CCGAGCCGTCCTGGTC
TaqMan® probe: 5'-FAM-AGTCGCAGGCGGCCACCGT-TAMRA
7.17.3 Preparation of primer/probes: Using a micropipettor with aerosol barrier tips, add PCR
grade water to the lyophilized primers and probe from the vendor to create stock
solutions of 500 uM primer and 100 uM probe and dissolve by extensive vortexing.
Pulse centrifuge to coalesce droplets. Store stock solutions at -20°C.
7.18 DNA extraction tube s
Note: It is recommended that tube preparation be performed in advance of water sampling and
DNA extraction procedures.
Prepare 1 tube for each sample, and 1 extra tube for every 6 samples (i.e., for method blank) and
minimum of 3 tubes per week for calibrator samples. Weigh 0.3 ± 0.01 g of glass beads (Section
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Method B
6.4) and pour into extraction tube. Seal the tube tightly, checking to make sure there are no beads
on the O-ring of the tube. Check the tube for proper O-ring seating after the tube has been closed.
Autoclave at 121°C (15 PSI) for 15 minutes.
7.19 Purified, RNA-free and spectrophotometrically quantified and characterized B. thetaiotaomicron
genomic DNA preparations used to generate a standard curve (see Section 11.2).
7.20 RNase A (e.g., Sigma Chemical #R-6513) or equivalent
7.20.1 Composition:
RNase A
Tris-Cl
NaCl
7.20.2 Dissolve 10 mg/mL pancreatic RNase A in 10 mM Tris-Cl (pH 7.5), 15 mM NaCl. Heat
to 100°C for 15 minutes. Allow to cool to room temperature. Dispense into aliquots and
store at - 20°C. For working solution, prepare solution in PCR-grade water at
concentration of 5 ug/uL.
7.21 DNA extraction kit (Gene-Rite #K102-02C-50 DNA-EZ® RW02 or equivalent)
8.0 Sample Collection, Handling, and Storage
8.1 Sampling procedures are briefly described below. Adherence to sample preservation procedures
and holding time limits is critical to the production of valid data. Samples not collected
according to these procedures should not be analyzed.
8.2 Sampling Techniques
Samples are collected by hand or with a sampling device if the sampling site has difficult access
such as a dock, bridge or bank adjacent to surface water. Composite samples should not be
collected, since such samples do not display the range of values found in individual samples. The
sampling depth for surface water samples should be 6-12 inches below the water surface. Sample
containers should be positioned such that the mouth of the container is pointed away from the
sampler or sample point. After removal of the container from the water, a small portion of the
sample should be discarded to provide head space for proper mixing before analyses.
8.3 Storage Temperature and Handling Conditions
Ice or refrigerate water samples at a temperature of <10°C during transit to the laboratory. Do
not freeze the samples. Use insulated containers to assure proper maintenance of storage
temperature. Take care that sample bottles are tightly closed and are not totally immersed in
water during transit.
8.4 Holding Time Limitations
Examine samples as soon as possible after collection. Do not hold samples longer than 6 hours
between collection and initiation of filtration. This section will be updated based on results of
holding time study.
9.0 Quality Control
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Method B
9.1 Each laboratory that uses Method B is required to operate a formal quality assurance (QA)
program that addresses and documents instrument and equipment maintenance and performance,
reagent quality and performance, analyst training and certification, and records sample storage
and retrieval. Additional recommendations for QA and quality control (QC) procedures for
microbiological laboratories are provided in Reference 17.2.
9.2 Media sterility check — The laboratory should test media sterility by incubating one unit (tube
or plate) from each batch of medium (5% BAP/ CMCB) as appropriate and observing for growth.
Absence of growth indicates media sterility. On an ongoing basis, the laboratory should perform
a media sterility check every day that samples are analyzed.
9.3 Method blank (water sample filtration blank) — Filter a 50 mL volume of sterile PBS before
beginning the sample filtrations. Remove the funnel from the filtration unit. Using two sterile or
flame-sterilized forceps, fold the filter on the base of the filtration unit and place it in an
extraction tube with glass beads as described in Section 7.20. Extract as in Section 11.5. Absence
of fluorescence growth curve during PCR analysis of these samples (reported as "0" on Smart
Cycler® and "undetermined" on ABI model 7900) indicates the absence of contaminant target
DNA (see Data Quality Acceptance, below). Prepare one method blank filter for every six
samples.
9.4 Positive controls — The laboratory should analyze positive controls to ensure that the method is
performing properly. Fluorescence growth curve (PCR amplification trace) with an appropriate
cycle threshold (CT) value during PCR indicates proper method performance. On an ongoing
basis, the laboratory should perform positive control analyses every day that samples are
analyzed. In addition, controls should be analyzed when new lots of reagents or filters are used.
9.4.1 Calibrator samples will serve as the positive control. Analyze as described in Section
11.0. Note: Calibrator samples contain the same amount of extraction buffer and starting
amount of Salmon DNA as the test samples, hence B. thetaiotaomicron calibrator DNA
extracts (Section 11.3) will be used as a positive control for both Bacteroidales and SPC
qPCR assays.
9.4.2 If the positive control fails to exhibit the appropriate fluorescence growth curve response,
check and/or replace the associated reagents, and reanalyze. If positive controls still fail
to exhibit the appropriate fluorescence growth curve response, prepare new calibrator
samples and reanalyze (see Section 9.7).
9.5 No template controls — The laboratory should analyze "No Template Controls" (NTC) to
ensure that the Master Mix PCR reagents are not contaminated. On an ongoing basis, the
laboratory should perform NTC analyses every day that samples are analyzed. If greater than
one-third of the NTC reactions for a PCR master mix exhibit true positive logarithmic
amplification traces with CT values below 45 (not from chemical degradation of probe with linear
kinetics that exhibit rising baseline) or if any one NTC reaction has a CT value lower than 35, the
analyses should be repeated with new Master Mix working stock preparations.
9.6 DNA standards and standard curves -Purified, RNA-free and spectrophotometrically
quantified B. thetaiotaomicron genomic DNA should be prepared as described in Section 11.2.
Based on reported values for it size, the weight of a single B. thetaiotaomicron genome can be
estimated to be -6.1 fg and there are six 16S rRNA gene copies per genome in this species
(http://cmr.jcvi.org/tigr-scripts/CMR/shared/Genomes.cgi). The concentration of 16S rRNA gene
copies per uL in the standard B. thetaiotaomicron genomic DNA preparation can be determined
from this information and from its spectrophotometrically determined total DNA concentration
by the formula:
10
-------
Method B
Concentration of Total DNA concentration (fg/uL) x 616SrRNAgene
16S rRNA gene copies copies
per uL =
6.7 fg/genome genome
A composite standard curve should be generated from multiple analyses of serial dilutions of this
DNA standard using the Bacteroidales primer and probe assay and subjected to linear regression
analysis as described in Section 12.2. From that point on, it is recommended that additional
standard curves be generated from duplicate analyses of these same diluted standard samples with
each new lot of TaqMan® master mix reagents or primers and probes to demonstrate comparable
performance by these new reagents. The r2 values from regressions of these curves should ideally
be 0.99 or greater. Comparable performance is assessed by their slopes and y-intercepts which
should be consistent with those from the initial composite analyses (e.g., within the 95%
confidence range of the average values). Note: The 95% confidence ranges for these parameters
in the initial composite standard curve can be generated using the Regression Analysis Tool
which can be accessed from the "Data Analysis" selection under the "Tools" menu in Excel.
Subsequent regressions can be performed by plotting the data using the Chart Wizard in Excel
and using the "add trend line" selection in the Chart menu and "display equation on chart"
selection under Options to obtain slope and y-intercept values as illustrated in Section 12.2.
In the event that the slope value from a subsequent standard curve regression is outside of the
acceptance range, the diluted standards should be re-analyzed. If this difference persists, new
working stocks of the reagents should be prepared and the same procedure repeated. If the
differences still persist, the amplification factor values used for calculations of target cell
numbers, as described in Section 8, should be modified based on the new slope values. If the
slopes are within acceptance range but Y-intercepts are not within acceptance range of this
previous average, new serial dilutions of the DNA standard should be prepared and analyzed as
described above.
9.7 Calibrator samples — The cell concentration of each cultured B. thetaiotaomicron stock
suspension used for the preparation of calibrator sample extracts should be determined as
described in Section 11.1. A minimum of nine calibrator sample extracts should initially be
prepared from three different freezer-stored aliquots of these stocks as described in Section 11.2.
Dilutions of each of these calibrator sample extracts equivalent to the anticipated dilutions of the
test samples used for analysis (e.g., 1, 5 and/or 25 fold) should be analyzed with the Bacteroidales
primer and probe assay. The average and standard deviation of the CT values from these
composite analyses should be determined. From that point on, a minimum of three fresh
calibrator sample extracts should be prepared from an additional frozen aliquot of the same stock
cell suspension at least weekly and preferably daily before analyses of each batch of test samples.
Dilutions of each new calibrator sample extract equivalent to the initial composite dilutions (e.g.,
1, 5 and/or 25 fold) should be analyzed using the Bacteroidales primer and probe assay. The
average CT value from these analyses should not be significantly different from the laboratory's
average values from analyses of the initial calibrator sample extracts from the same stock cell
suspension (i.e., not greater than three standard deviations). If these results are not within this
acceptance range, new calibrator extracts should be prepared from another frozen aliquot of the
same stock cell suspension and analyzed in the same manner as described above. If the results are
still not within the acceptance range, the reagents should be checked by the generation of a
standard curve as described in Section 9.6.
9.8 Salmon DNA Sample Processing Control (SPC) sequence analyses — While not essential, it
is good practice to routinely prepare and analyze standard curves from serial dilutions of Salmon
-------
Method B
DNA working stocks in a manner similar to that described for the B. thetaiotaomicron DNA
standards in Section 9.6. At this time rRNA gene operon copy numbers per genome have not been
reported in the literature for the salmon species O. keta. Therefore log-transformed total DNA
concentration values or dilution factor values can be substituted for target sequence copy numbers
as the x-axis values in these plots and regression analyses.
In general, target DNA concentrations in test samples can be calculated as described in Section
12. However, the Salmon DNA PCR assay results for each test sample's 5 fold dilution should
be within 3 CT units of the mean of the 5 fold diluted calibrator (and/or method blank) sample
results. Higher CT values may indicate significant PCR inhibition or poor DNA recovery
possibly due to physical, chemical, or enzymatic degradation. Repeat the Bacteroidales and
Salmon DNA PCR assays of any samples whose 5 fold dilution exhibits a Salmon DNA PCR
assay CT value greater than 3 CT units higher than the mean of the calibrator sample results using
a 5 fold higher dilution (net dilution: 25 fold) of the extracts. The Bacteroidales PCR result from
assaying the original 5-fold dilution of the sample can be accepted if its Salmon DNA assay CT
value is lower than that of the corresponding 25 fold dilution of the sample. This pattern of
results is indicative of poor recovery of total DNA in the extract rather than PCR inhibition. The
poor DNA recovery is compensated for by the calculation method. Contrarily, if the Salmon
PCR assay CT value of the 25-fold dilution of the sample is lower than that of the 5 fold dilution
of the sample, then the Bacteroidales PCR assay result from the 25 fold dilution of the sample is
considered more accurate. However the Bacteroidales PCR results should be reported as
questionable if the Salmon DNA assay's result is still not within 3 CT units of the mean CT result
of the 25 fold dilution of the three calibrators.
10.0 Calibration and Standardization of Method-Related Instruments
10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to
ensure operation within stated limits.
10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the
requirements of NIST Monograph SP 250-23. Check columns for breaks.
10.3 Spectrophotometer should be calibrated each day of use using OD calibration standards between
0.01 - 0.5. Follow manufacturer instructions for calibration.
10.4 Micropipettors should be calibrated at least annually and tested for accuracy on a weekly basis.
Follow manufacturer instructions for calibration.
10.5 Follow manufacturer instructions for calibration of real-time PCR instruments.
11.0 Procedure
Note: B. thetaiotaomicron cell suspensions (Section 11.1), and B. thetaiotaomicron DNA
standards (Section 11.2) may be prepared in advance. Calibrator samples (Section 11.3) should
be prepared at least weekly.
11.1 Preparation of B. thetaiotaomicron cell suspensions for DNA standards and calibrator samples
11.1.1 Thaw a B. thetaiotaomicron (ATCC #29741) stock culture (Section 7.8) and streak for
isolation on CDC anaerobe 5% sheep blood agar (BAP) plates. Incubate plates at 35.0°C
± 0.5°C for 24 ± 2 hours under anaerobic conditions.
11.1.2 Pick an isolated colony of B. thetaiotaomicron from the BAP plates and suspend in 1 mL
of sterile phosphate buffered saline (PBS) and vortex.
12
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Method B
11.1.3 Use 10 uL of the 1-mL suspension of B. thetaiotaomicron to inoculate a 10-mL CMCB
tube. Place the inoculated tube and one uninoculated tube (sterility check) in an anaerobe
chamber with a Gas Pak and incubate at 35.0°C ± 0.5°C for 24 ±2 hours. Note: It is
advisable to verify that the selected colony is Bacteroides by using biochemical test strips
or individual biochemical tests.
11.1.4 After incubation remove septum and transfer liquid to a sterile tube by pipetting. While
pipetting the suspension use extreme care to remove as much liquid as possible without
siphoning any of the chopped meat.
11.1.5 Centrifuge the CMCB containing B. thetaiotaomicron for 5 minutes at 6000 x g.
11.1.6 Aspirate the supernatant and resuspend the cell pellet in 10 mL PBS.
11.1.7 Repeat the two previous steps twice and suspend final B. thetaiotaomicron pellet in 5 mL
of sterile PBS. Label the tube as B. thetaiotaomicron undiluted stock cell suspension,
noting cell concentration after determination with one of the following steps.
11.1.8 Determination of calibrator sample cell concentrations based on one of the three options
below.
• Option 1: Spectrophotometric absorbance - Remove three 0.1-mL aliquots of
undiluted cell suspension and dilute each with 0.9 mL of PBS (10"1 dilution). Read
absorbance at 595 nm in spectrophotometer against PBS blank (readings should
range from 0.05 to 0.3 OD). Calculate cells/mL (Y) in undiluted cell suspension
using the formula below where X is the average 595 nm spectrophotometer reading.
Y = (1 x 109 cells / mL x X) / 0.19
• Option 2: Hemocvtometer counts - Serially dilute 10 uL of undiluted cell suspension
with PBS to 10"1, 10"2, and 10"3 dilutions and determine cell concentration of 10"2 or
10"3 dilutions in a hemocytometer or Petroff Hauser counting chamber under
microscope.
• Option 3: plating - Note: BAP plates should be prepared in advance if this option is
chosen. For enumeration of the B. thetaiotaomicron undiluted cell suspension, dilute
and inoculate according to the following.
A) Mix the B. thetaiotaomicron undiluted cell suspension by shaking or vortexing
the 5 mL tube a minimum of 25 times. Use a sterile pipette to transfer 1.0 mL of
the undiluted cell suspension to 99 mL of sterile PBS use care not to aspirate
any of the particulates in the medium, cap, and mix by vigorously shaking the
bottle a minimum of 25 times. This is cell suspension dilution "A". A 1.0-mL
volume of dilution "A" is 10"2 mL of the original undiluted cell suspension.
B) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "A" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is cell suspension dilution "B". A 1.0-mL volume of dilution "B" is
10"3 mL of the original undiluted cell suspension.
C) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "B" to 99
mL of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of
25 times. This is cell suspension dilution "C". A 1.0-mL volume of dilution
"C" is 10"4 mL of the original undiluted cell suspension.
D) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "C" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
13
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Method B
times. This is cell suspension dilution "D". A 1.0-mL volume of dilution "D" is
10"5 mL of the original undiluted cell suspension.
E) Use a sterile pipette to transfer 11.0 mL of cell suspension dilution "D" to 99 mL
of sterile PBS, cap, and mix by vigorously shaking the bottle a minimum of 25
times. This is cell suspension dilution "E". A 1.0-mL volume of dilution "E" is
10"6 mL of the original undiluted cell suspension.
F) Prepare BAP (Section 7.6). Ensure that agar surface is dry. Note: To ensure that
the agar surface is dry prior to use, plates should be made several days in
advance and stored inverted at room temperature or dried using a laminar-flow
hood.
G) Each of the following will be conducted in triplicate, resulting in the evaluation
of nine spread plates:
• Pipet 0.1 mL of dilution "C" onto surface of BAP plate [10"5 mL (0.00001) of
the original cell suspension].
• Pipet 0.1 mL of dilution "D" onto surface of BAP plate [10"6 mL (0.000001)
of the original cell suspension].
• Pipet 0.1 mL of dilution "E" onto surface of BAP plate [10"7 mL (0.0000001)
of the original cell suspension].
H) For each spread plate, use a sterile bent glass rod or spreader to distribute
inoculum over surface of medium by rotating the dish by hand or on a rotating
turntable.
Allow inoculum to absorb into the medium completely
I)
J)
Invert plates and incubate in an anaerobe chamber at 35.0°C ± 0.5°C for 24 ± 4
hours.
K) Count and record number of colonies per plate. Refer to the equation below for
calculation of undiluted cell suspension concentration.
ited
+ CFUn
Vi + V-)
Where:
CFU/mLundliuted -
CPU
V
B. thetaiotaomicron CFU/mL in undiluted cell suspension
number of colony forming units from BAP plates yielding
counts within the ideal range of 30 to 300 CPU per plate
volume of undiluted sample in each BAP plate yielding
counts within the ideal range of 30 to 300 CPU per plate
number of plates with counts within the ideal range
14
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Method B
Table 1. Example Calculations of B. thetaiotaomicron Undiluted Cell Suspension Concentration
Examples
Example 1
Example 2
CPU / plate (triplicate analyses) from
BAP plates
10'5 mL plates
275, 250, 301
TNTCb, TNTC,
TNTC
10'6 mL plates
30, 10, 5
TNTC, 299, TNTC
10~7 mL plates
0, 0, 0
12, 109, 32
B. thetaiotaomicron CPU / mL in
undiluted
suspension3
(275+250+30) /(10-5+10-5+10-6) =
5557 (2.1 x10'S) = 26,428,571 =
2.6x107CFU/mL
(299+1 09+32) /(10'6+10-7+10-7) =
440 / (1 .2 x 1 0'6) =366,666,667 =
3.7x108CFU/mL
Cell concentration is calculated using all plates yielding counts within the ideal range of 30 to 300 CPU per plate
b Too numerous to count
11.1.9 Divide remainder of undiluted cell suspension (approximately 5 mL) into 6 x 0.5 mL
aliquots for DNA standard preparations and 100-200 x 0.01 mL (10 ^L) aliquots for
calibrator samples and freeze at -20°C. Note: Cell suspension should be stirred while
aliquoting. It is also recommended that separate micropipettor tips be used for each 10
jwL aliquot transfer and that the volumes in each tip be checked visually for consistency.
11.2 Preparation of B. thetaiotaomicron genomic DNA standards
11.2.1 Remove two 0.5 mL undiluted B. thetaiotaomicron cell suspensions (Section 11.1.8)
from freezer and thaw completely.
11.2.2 Transfer cell suspensions to extraction tubes with glass beads.
11.2.3 Tightly close the tubes, making sure that the O-rings are seated properly.
11.2.4 Place the tubes in bead beater and shake for 60 seconds at the maximum rate (5000 rpm).
11.2.5 Remove the tubes from the bead beater and centrifuge at 12,000 x g for one minute to
pellet the glass beads and debris.
11.2.6 Using a 200 uL micropipettor, transfer 350 uL of supernatants to sterile 1.7 mL
microcentrifuge tubes. Recover supernatants without disrupting the glass beads at the
tube bottom.
11.2.7 Centrifuge crude supernatants at 12,000 x g for 5 minutes and transfer 300 uL of clarified
supernatant to clean, labeled 1.7 mL low retention microcentrifuge tubes, taking care not
to disturb the pellet.
11.2.8 Add 1 uL of 5 ug/uL RNase A solution to each clarified supernatant, mix by vortexing
and incubate at 37°C for 1 hour.
Note: Sections 11.2.9 - 11.2.15 may be substituted with an optional method if a DNA
purification procedure is chosen other than the DNA-EZ purification kit. In such a case,
manufacturer instructions should be followed rather than these steps. Continue onward
from Section 11.2.16.
11.2.9 Add 0.6 mL of binding buffer solution from a DNA-EZ purification kit to each of the
RNase A-treated extracts and mix by vortexing. Note: In general, a minimum
concentration of 5 x 108 cells is required for this step.
15
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Method B
11.2.10 If using the DNA-EZ purification kit, perform the following steps. Insert one
DNAsure™ column from the DNA-EZ purification kit into a collection tube (also
provided with kit) for each of the two extracts. Transfer the extract and binding buffer
mixtures from Section 11.2.9 to a DNAsure™ column and collection tube assembly
and centrifuge for 1 min at 12,000 x g.
11.2.11 Transfer each of the DNAsure™ columns to new collection tubes. Discard previous
collection tubes and collected liquid.
11.2.12 Add 500 uL EZ-Wash Buffer from the DNA-EZ purification kit to each of the
DNAsure™ columns and centrifuge at 12,000 x g for 1 minute. Discard the liquid in
the collection tube.
11.2.13 Repeat Section 11.2.12.
11.2.14 Transfer each of the DNAsure™ columns to a clean, labeled 1.7-mL low retention
microcentrifuge tube and add 50 uL of DNA elution buffer to each column.
Centrifuge for 30 seconds at 12,000 x g. Repeat this procedure again to obtain a total
DNA eluate volume of-100 uL from each column.
11.2.15 Pool the two eluates to make a total volume of approximately 200 uL.
11.2.16 Transfer entire purified DNA eluate volume from each column to a clean and sterile
microcuvette for UV spectrophotometer and read absorbance at 260 and 280 nM.
(Note: the cuvette should be blanked with DNA elution buffer before reading sample.)
If necessary, the sample may be diluted with elution buffer to reach minimum volume
that can be accurately read by the spectrophotometer (see manufacturer's
recommendation) however, this may reduce the DNA concentration to a level that can
not be accurately read by the spectrophotometer. If available, readings can be taken of
2 uL aliquots of the sample with aNanoDrop™ Spectrophotometer.
11.2.17 Sample is acceptable as a standard if ratio of OD26o/OD28o readings is >to 1.75.
11.2.18 Calculate total DNA concentration in sample by formula:
OD260 reading x 50 ng/uL DNA/1(OD260)
11.2.19 Transfer sample back to labeled 1.7 mL non-retentive microcentrifuge tube and store
at -20°C.
11.3 Extraction of B. thetaiotaomicron calibrator samples
11.3.1 A minimum of three calibrator extracts should be prepared during each week of analysis.
Note: To prevent contamination of water sample filtrates and filter blanks, this procedure
should be performed at a different time, and, if possible, in a different work station than
the procedures in Sections 11.1 and 11.2 above and Section 11.5 below.
11.3.2 Remove one tube containing a 10 uL aliquot of B. thetaiotaomicron undiluted stock cell
suspension (Section 11.1.8) from the freezer and allow to thaw completely on ice.
Note: If using BioBalls for calibrators add a single BioBall™ to each of 3, 100 mL sterile
PBS blanks, filter (Section 11.4), and extract according to (Section 11.5).
11.3.3 While cell stock is thawing, using sterile (or flame-sterilized) forceps, place one
polycarbonate filter (Section 6.14) in an extraction tube with glass beads. Prepare one
filter for each sample to be extracted in this manner.
16
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Method B
11.3.4 Dispense 590 uL of Salmon DNA/extraction buffer (Section 7.12) into three extraction
tubes with glass beads and filters. Prepare one tube for each of the three calibrator
samples to be extracted in this manner. Label tubes appropriately.
11.3.5 When B. thetaiotaomicron suspension has thawed, transfer 990 uL AE buffer (Section
7.11) to the 10 uL B. thetaiotaomicron stock cell tube and mix thoroughly by vortexing.
Pulse microcentrifuge tube briefly (1-2 sec.) to coalesce droplets in tube.
11.3.6 Immediately after vortexing the B. thetaiotaomicron suspension, spot 10 uL onto the
polycarbonate filter in a calibrator sample tube.
11.3.7 Tightly close the tube, making sure that the O-ring is seated properly.
11.3.8 Repeat Sections 11.3.6 and 11.3.7 for the other two filters to prepare three calibrator
samples with B. thetaiotaomicron.
11.3.9 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate
(5000 rpm).
11.3.10 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for one
minute to pellet the glass beads and debris.
11.3.11 Using a 200 uL micropipettor, transfer the crude supernatant to the corresponding
labeled sterile 1.7-mL microcentrifuge tube. Transfer 400 uL of supernatant without
disrupting the debris pellet or glass beads at the tube bottom. Note: The filter will
normally remain intact during the bead beating and centrifugation process. Generally,
400 uL of supernatant can be easily collected. Collect an absolute minimum of 100
uL of supernatant.
11.3.12 Centrifuge at 12,000 x g for 5 minutes and transfer clarified supernatant to a clean,
labeled 1.7 mL tube, taking care not to disturb the pellet. Note: Cell pellet may not be
visible in calibrator samples.
11.3.13 Label the tubes as undiluted or Ix B. thetaiotaomicron calibrator extracts. Label
additional 1.7 mL tubes for 5 and 25 fold dilutions. In appropriately labeled tubes,
using a micropipettor, add a 50 uL aliquot of each Ix B. thetaiotaomicron calibrator
extract and dilute each with 200 uL AE buffer (Section 7.11) to make 5 fold dilutions.
In appropriately labeled tubes using a micropipettor, add a 50 uL aliquot of each 5
fold dilution and dilute each with 200 uL AE buffer to make 25 fold dilutions. Store
all diluted and undiluted extracts in refrigerator.
11.3.14 If the extracts are not analyzed immediately, refrigerate. For long term storage, freeze
at -80°C.
11.4 Water sample filtration
Note: It is required that one water sample filtration blank (method blank) be prepared for every 6
water samples (Section 9.3) analyzed by the same procedure.
11.4.1 Place a polycarbonate filter (Section 6.16) on the filter base, and attach the funnel to the
base so that the membrane filter is now held between the funnel and the base.
11.4.2 Shake the sample bottle vigorously 25 times to distribute the bacteria uniformly, and
measure 100 mL of sample into the funnel.
11.4.3 Filter 100 mL of water sample. After filtering the sample, rinse the sides of the funnel
with 20-30 mL of sterile PBS (Section 7.4) and continue filtration until all liquid has been
pulled through the filter. Turn off the vacuum and remove the funnel from the filter base.
17
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Method B
11.4.4 Label an extraction tube with glass beads (Section 7.19) to identify water sample.
Leaving the filter on the filtration unit base, fold into a cylinder with the sample side
facing inward, being careful to handle the filter only on the edges, where the filter has not
been exposed to the sample. Insert the rolled filter into the labeled extraction tube with
glass beads. Prepare one filter for each sample filtered in this manner.
11.4.5 Cap the extraction tube. Tubes may be frozen at -20°C or -80°C until analysis.
11.5 DNA extraction of water sample filtrates and method blanks
11.5.1 Using a 1000 uL micropipettor, dispense 590 uL of the Salmon DNA/extraction buffer
(Section 7.13) to each labeled extraction tube with glass beads containing water sample
or method blank filters from Section 11.4.4. Extract the method blank last.
11.5.2 Tightly close the tubes, making sure that the O-ring is seated properly.
11.5.3 Place the tubes in the mini bead beater and shake for 60 seconds at the maximum rate
(5000 rpm).
11.5.4 Remove the tubes from the mini bead beater and centrifuge at 12,000 x g for 1 minute to
pellet the glass beads and debris. Note: To further prevent contamination, a new pair of
gloves may be donned for steps 11.5.5, 11.5.6, and 11.5.7 below.
11.5.5 Using the 200 uL micropipettor, transfer 400 uL of the supernatant to a corresponding
labeled sterile 1.7-mL microcentrifuge tube, taking care not to pick up glass beads or
sample debris (pellet). Note: The filter will normally remain intact during the bead
beating and centrifugation process. Generally, 400 uL of supernatant can easily be
collected. Collect an absolute minimum of 100 \\L of supernatant. Recover the method
blank supernatant last.
11.5.6 Centrifuge crude supernatant from Section 11.5.5 for 5 minutes at 12,000 * g. Transfer
350 uL of the clarified supernatant to another 1.7-mL tube, taking care not to disturb
pellet. Note: Pellet may not be visible in water samples. Recover the method blank
supernatant last.
11.5.7 Label the tubes from Section 11.5.6 as undiluted or Ix water sample extracts with sample
identification. These are the water sample filter extracts. Also label tubes for method
blanks. Label additional 1.7 mL tubes for 5 and 25 fold dilutions. In appropriately
labeled tubes, using a micropipettor, add a 50 uL aliquot of each Ix water sample extract
and dilute each with 200 uL AE buffer (Section 7.11) to make 5 fold dilutions. In
appropriately labeled tubes using a micropipettor, add a 50 uL aliquot of each 5 fold
dilution and dilute each with 200 uL of AE buffer to make 25 fold dilutions. Dilute the
method blank supernatant last.
11.5.8 Store all diluted and undiluted extracts in refrigerator. Note: Use of 5 fold diluted
samples for analysis is currently recommended if only one dilution can be analyzed.
Analyses of undiluted water sample extracts have been observed to cause a significantly
higher incidence of PCR inhibition while analyses of 25 fold dilutions may unnecessarily
sacrifice sensitivity.
11.5.9 If the extracts are not analyzed immediately, refrigerate. For long term storage, freeze at
-80°C.
11.6 Preparation of qPCR assay mix
11.6.1 To minimize environmental DNA contamination, routinely treat all work surfaces with a
10% bleach solution, allowing the bleach to contact the work surface for a minimum of
18
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Method B
15 minutes prior to rinsing with sterile water. If available, turn on UV light for 15
minutes. After decontamination, discard gloves and replace with new clean pair.
11.6.2 Remove primers and probe stocks from the freezer and verify that they have been diluted
to solutions of 500 uM primer and 100 uM probe.
11.6.3 Prepare working stocks of B. thetaiotaomicron, and Salmon DNA (Sketa 22)
primer/probe mixes by adding 10 uL of each B. thetaiotaomicron or Salmon DNA (Sketa
22) primer stock and 4 uL of respective probe stock to 676 uL of PCR grade water, and
vortex. Pulse centrifuge to create a pellet. Use a micropipettor with aerosol barrier tips
for all liquid transfers. Transfer aliquots of working stocks for single day use to separate
tubes and store at 4° C.
11.6.4 Using a micropipettor, prepare assay mix of the B. thetaiotaomicron, and Salmon DNA
(Sketa 22) reactions in separate, sterile, labeled 1.7 mL microcentrifuge tubes as
described in Table 2.
Table 2. PCR Assay Mix Composition
Reagent
Sterile H2O
BSA
TaqMan® master mix
Primer/probe working stock solution
Volume/Sample (multiply by # samples to be analyzed
per day)
1.5|JL
2.5 pi
12.5 pi
3.5 pi
16 samples plus and 1 extra - see Section 11.7
Note: This will give a final concentration of 1 uM of each primer and 80 nM of probe in the
reactions. Prepare sufficient quantity of assay mix for the number of samples to be analyzed per
day including calibrators and negative controls plus at least two extra samples. Prepare assay
mixes each day before handling of DNA samples.
11.6.5 Vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.
11.7 ABI 7900 and ABI 7500 (non-Fast) qPCR assay preparation (Reference 17.1)
Transfer 20 uL of mastermix containing Bacteroidales primers and probe to wells of a 96-well
PCR reaction tray equal to number of samples to be analyzed including calibrator and negative
control samples. (Note: The same tip can be used for pipetting multiple aliquots of the same
assay mix as long as it doesn't make contact with anything else).
Example: For the analysis of 18 recreational water samples, 51 wells will require the addition of
assay mix with B. thetaiotaomicron primers and probe as follows: 18 samples, two replicates each
(36), 3 method blanks, two replicates each (6), 3 no template controls, one replicate each (3), and
3 calibrators, 2 replicates each (6) = 51 wells.
11.7.1 Transfer 20 uL of mastermix containing B. thetaiotaomicron primers and probe to wells
of a 96 well PCR reaction tray equal to number of samples to be analyzed including
calibrator and negative control samples. Pipette into the center of the wells, taking care to
not touch the well walls with the pipette tip. (Note: The same tip can be used for pipetting
multiple aliquots of the same assay mix as long as it doesn't make contact with anything
else).
11.7.2 When all wells are loaded, cover tray loosely with aluminum foil or plastic wrap and
transfer to refrigerator or directly to the PCR preparation station used for handling DNA
19
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Method B
samples (Section 6.1). Note: All aliquoting of assay mixes to reaction trays must be
performed each day before handling of DNA samples.
11.7.3 Transfer 5 uL each of the diluted (or undiluted) DNA extracts of method blanks and
water samples (Section 11.5.7), and then corresponding dilutions of calibrator samples
(Section 11.3.13), to separate wells of the PCR reaction tray containing Bacteroidales
assay mix. Note: Record positions of each sample.
11.7.4 Transfer 5 uL each of the diluted (or undiluted) DNA extracts of method blanks and
water samples (Section 11.5.7), and corresponding diluted calibrator samples (Section
11.3.13) to separate wells of the PCR reaction tray containing Salmon DNA assay mix.
Record positions of each sample.
11.7.5 Transfer 5 uL aliquots of AE buffer to wells of PCR reaction tray containing B.
thetaiotaomicron master mix that are designated as no-template controls. Record
positions of these samples.
11.7.6 Tightly cap wells of PCR reaction tray containing samples or cover tray and seal tightly
with optical adhesive PCR reaction tray.
11.7.7 Run reactions in ABI 7900 or ABI 7500 (non-Fast) sequence detector. For platform-
specific operation see Appendix A.
11.8 Smart Cycler® qPCR assay preparation
11.8.1 Label 25 uL Smart Cycler® tubes with sample identifiers and assay mix type (see
Section 11.8.8 for examples) or order tubes in rack by sample number and label rack with
assay mix type. It is recommended that the unloaded open Smart Cycler® tubes be
irradiated under ultraviolet light in a PCR cabinet for 15 minutes. Using a micropipettor,
add 20 uL of the Bacteroidales assay mix (Section 11.6.5) to labeled tubes. Avoid
generating air bubbles, as they may interfere with subsequent movement of the liquid into
the lower reaction chamber. The same tip can be used for pipetting multiple aliquots of
the same assay mix as long as it doesn't make contact with anything else. Repeat
procedure for Salmon DNA (Sketa 22) assay mix.
11.8.2 Add 5 uL of AE buffer to no-template control tubes and close tubes tightly.
11.8.3 Close the other PCR tubes loosely and transfer to refrigerator or directly to the PCR
preparation station used for handling DNA samples (Section 6.1). Note: All aliquoting of
assay mixes to reaction tubes must be performed each day before handling of DNA
samples.
11.8.4 Transfer 5 uL each of the diluted (or undiluted) DNA extracts of method blanks and
water samples (Section 11.5.7), and then corresponding dilutions of calibrator samples
(Section 11.3.13) to tubes containing B. thetaiotaomicron and Salmon DNA (Sketa 22)
mixes. Close each tube tightly after adding sample. Load the method blank PCR assays
last. Label the tube tops as appropriate.
11.8.5 When all Smart Cycler® tubes have been loaded, place them in a Smart Cycler®
centrifuge, and spin for 2-4 seconds.
11.8.6 Inspect each tube to verify that the sample has properly filled the lower reaction chamber.
A small concave meniscus may be visible at the top of the lower chamber, but no air
bubbles should be present. (If the lower chamber has not been properly filled, carefully
open and reclose the tube, and re-centrifuge). Transfer the tubes to the thermocycler.
11.8.7 For platform-specific operation see Appendix B.
11.8.8 Suggested sample analysis sequence for Smart Cycler®
20
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Method B
Example: For analyses on a single 16-position Smart Cycler®, calibrator samples and
water samples will need to be analyzed in separate runs and a maximum of 6 water
samples (or 2 replicates of 3 samples) can be analyzed per run, as described in Tables 3
and 4, below.
Table 3. Calibrator PCR Run -14 Samples
Sample Description*
3 Calibrators * 2 replicates (1, 5, or 25 fold dilution )
3 Calibrators * 2 replicates (1, 5, or 25 fold dilution )
No template controls (reagent blanks)
Quantity
6
6
2
PCR Assay Master Mix
6. thetaiotaomicron
Salmon DMA
B. thetaiotaomicron
* Diluted equivalently to the water samples
Table 4. Water Sample PCR Run -14 Samples
Sample Description*
Water samples, (1, 5, or 25 fold dilution )
Method blank, (1, 5, or 25 fold dilution )
Water samples, (1, 5, or 25 fold dilution )
Method blank, (1, 5, or 25 fold dilution )
Quantity
6
1
6
1
PCR Assay Master Mix
B. thetaiotaomicron
B. thetaiotaomicron
Salmon DMA
Salmon DMA
* Use of five-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed.
Analyses of undiluted water sample extracts have been observed to cause a significantly higher incidence of PCR
inhibition while 25 fold dilutions analyses may unnecessarily sacrifice sensitivity.
12.0 Data Analysis and Calculations
12.1 Overview: This section describes a method for determining the ratio of the target sequence
quantities recovered from a test (water filtrate) sample compared to those recovered from
identically extracted calibrator samples using an arithmetic formula, referred to as the AACT
comparative cycle threshold calculation method. The AACT relative quantitation method also
normalizes these ratios for differences in total DNA recovery from the test and calibrator samples
using qPCR analysis CT values for a reference sequence provided by the SPC DNA. These ratios
are converted to absolute measurements of total target sequence quantities recovered from the test
samples by multiplying them by the average total number of target sequences that are normally
recovered from a constant number of target organisms that are added to all calibrator samples.
The complete procedure for determining target sequence quantities in water samples is detailed
below.
12.2 Generation of CT value vs. target sequence number standard curve: Three replicate serial
dilutions of a DNA standard, prepared as described in Section 9.6 should be prepared to give
concentrations of 4 x 104, 4 x 103, 4 x 102, 2 x 102 and 1 x 102 16S rRNA gene sequences per 5 uL
(the standard sample volume added to the PCR reactions) and the replicates of each dilution
pooled. Note: A procedure for the determinations of target sequence concentrations in the DNA
standard is also provided in Section 9.6.
Aliquots of each of these dilutions should be stored at 4°C in low retention microcentrifuge tubes
and can be reused for repeated qPCR analyses. QPCR analyses of these diluted standards using
the Bacteroidales primer and probe assay should be performed at least three separate times in
duplicate. CT values from these composite analyses should be subjected to regression analysis
21
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Method B
against the loglO-transformed target sequence numbers per reaction as described in Section 9.6
with example results illustrated in Figure 1.
234
Log target sequences per reaction
Figure 1: Example plot and regression analysis of qPCR analysis cycle threshold values vs. log target
sequences per reaction.
Amplification factors (AF) used for subsequent comparative cycle threshold calculations (Section
12.4) can be calculated from the slope value of this curve by the formula AF = 10A(1 / (-)slope
value). An example calculation using the slope value from the example regression is shown
below:
AF=10A(1 73.4777) = 1.94
12.3 Calculation of average target sequence recovery in calibrator sample extracts: A minimum
of nine calibrator sample extracts should initially be prepared from at least three different freezer-
stored aliquots of each cultured B. thetaiotaomicron stock suspension that is prepared as
described in Section 11.1. Dilutions of each of these calibrator sample extracts equivalent to the
anticipated dilutions of the test samples used for analysis (e.g., 1,5 and/or 25 fold) should be
analyzed with the Bacteroidales primer and probe assay. The average CT value from these
analyses should be interpolated on the standard curve generated from the DNA standard (Section
12.2) to determine the average number of target sequences per 5 uL of extract used in the
reactions. An example calculation using an average calibrator extract Cr value of 25.21 is shown
below:
Average calibrator target sequences/5 uL extract = 10A((25.21-38.44) / -3.477) = 6382
Note: Four places should be kept from this calculation for the following calculation (i.e.,
6382.6983). Dividing this value by 5 gives the average calibrator target sequences/uL extract
which can be multiplied by the total volume of the extract at the applicable dilution level (e.g.,
600 uL of original extract volume x 5 = 3000 uL for a 5 fold diluted sample) to determine the
average total quantity of target sequences recovered in the calibrator sample extracts. An
example of this calculation using the average calibrator target sequences/reaction value
determined immediately above is shown below:
Average target sequences = 6382 target sequences x 3000 uL total extract volume
22
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Method B
Calibrator extract 5 \\L extract
= 3,829,619
12.3.1 Calculation of average target sequence recovery per Bacteroidales cell in calibrator
sample extracts (optional): In previous studies, measurements of recreational water
quality by the qPCR method have been reported as Bacteroidales calibrator cell
equivalents (Reference 17.7). Calculations performed to obtain this reporting unit are
identical to those described in Section 12.4 except that the ratios of target sequences
obtained as described in Sections 12.4.1 - 12.4.4 are multiplied by the estimated
quantities of Bacteroidales cells added to the calibrator samples rather than by the
average target sequences recovered per calibrator extract as described in Section 12.4.5.
While the use of this reporting unit is no longer recommended because of the false
impression it creates concerning the cell concentration detection limit of the qPCR
method, it still may be of value for comparing previous results with those of future
studies.
A prerequisite for making such comparisons is to determine that the ratio of the numbers
of target sequences recovered in calibrator sample extracts to the numbers of
Bacteroidales cells added to these samples is consistent in different studies. For the
purpose of determining this ratio, it is recommended that the cell concentrations of the
cultured B. thetaiotaomicron stock suspension used for the preparation of calibrator
samples in each laboratory be determined by at least two of the three alternative methods
described in Section 11.1 to establish the degree of agreement between these enumeration
methods. The recommended quantity of cells that are added to each calibrator sample is
100,000. Dividing the average target sequences recovered per calibrator extract
(determined as described in Section 12.3) by this number provides the ratio of target
sequence numbers to cell numbers. An example of this calculation using the average
target sequences/ calibrator extract value determined in Section 12.3 is shown below:
Ratio of target sequence numbers to cell numbers = 3,829,619 / 100,000 = 38.29
12.4 Calculation of target sequence quantities in test samples: A minimum of three fresh calibrator
samples should be extracted and analyzed at least on a weekly basis and preferably on a daily
basis in association with each batch of water sample filtrates. QC analysis of the analysis results
from these calibrator extracts should be performed as described in Section 9.7. C T values from
the Bacteroidales target sequence and salmon DNA Sample Processing Control (SPC) qPCR
assays for both the calibrator and test samples are used in the AACT comparative cycle threshold
calculation method to determine the ratios of target sequences in the test and calibrator sample
extracts and these ratios are converted to absolute measurements of total target sequence
quantities recovered from the test samples as specified below and illustrated in Table 5.
12.4.1 Subtract the SPC assay CT value (CT,spc) from the target assay CT value (CT,target) for each
calibrator sample extract to obtain ACT value and calculate the average ACT value for
these calibrator samples.
12.4.2 Subtract the SPC assay CT value (CT,spc) from the target assay CT value (CT,target) for each
water sample filtrate extract to obtain ACT values for each of these test samples. Note: If
multiple analyses are performed on these samples, calculate the average ACT value.
12.4.3 Subtract the average ACT value for the calibrator samples from the ACT value (or average
ACT value) for each of the test samples to obtain AACT values.
23
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Method B
12.4.4 Calculate the ratio of the target sequences in the test and calibrator samples using the
formula: AFA("AACT), where AF = amplification factor of the target organism qPCR assay,
determined as described in Section 12.2.
12.4.5 Multiply the ratio of the target sequences in the test and calibrator samples by the average
target sequences/calibrator extract, determined as described in Section 12.2, to determine
absolute numbers of total target sequences/ extract for each of the test samples. Note:
This calculation can be applied without modification to the analyses of diluted extracts if
both the test sample and calibrator extracts are equally diluted and equal volumes of
diluted extracts are analyzed.
Table 5. Exam
ile Calculations (Amplification factor = 1.94)
Target
sequences
in Sample
3,829,619
Unknown
Sample
Type
Calibrator
Test
Cj.target
25.21
32.53
CT.SPC
30.45
30.65
ACT
-5.24
1.88
AACT
—
7.12
Measured Target Sequences in
Test Sample Extract
(1. 94 -iiCTxavg. target
sequences/calibrator)
—
0.0089x3,829,619 = 34,198
12.4.6 The geometric mean of the measured target sequences and associated coefficients of
variation in multiple water samples can be determined from individual sample CT values
using the following procedure:
12.4.6.1
12.4.6.2
12.4.6.3
Use ACT value for each individual water sample extract and the mean
calibrator ACT value to calculate the measured target sequence numbers in
each water sample extract, as described in Section 12.4.
Calculate the logio of the measured target sequence numbers in each water
sample (log N)
Calculate the mean (M) and standard deviation (S) from the values of log N
obtained in the previous step for all of the water sample extracts.
Calculate the geometric mean as 10M.
The implied coefficient of variation (CV) is calculated, based on the log
normal distribution, as the square root of iov/0434 - 1, where V = S2.
12.5 Reporting Results: Where possible, duplicate analyses should be performed on each sample.
Report the results as Bacteroidales (16S rRNA gene) target sequences per volume of water
sample filtered.
12.4.6.4
12.4.6.5
13.0 Sample Spiking Procedure
[This section will be updated after validation study]
24
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Method B
14.0 Method Performance
[This section will be updated after validation study]
14.1 Accuracy (Bias)
The 16S rRNA gene of Bacteroidales, which contains the target sequence amplified and detected
by the primers and probe of the Bacteroidales qPCR assay, is present in multiple copies in the
genome of the Bacteroidales order. The number of 16S rRNA gene copies per genome has not
been ascertained for all of the Bacteroidales order which the Bacteroidales qPCR assay can
amplify and detect. Hence, the use of B. thetaiotaomicron cells as a calibrator for relative
quantitation purposes and B. thetaiotaomicron DNA as a standard for absolute quantitation
purposes creates an inherent bias potentially affecting the accuracy of the quantitation depending
on the species composition of the Bacteroidales present in a water sample.
The Bacteroidales qPCR method makes the assumption that the Bacteroidales cells present in the
water sample contain the same number of genomes and 16S rRNA gene copies as the B.
thetaiotaomicron calibrator cells which have been grown in culture media to a late-log or
stationary phase in batch culture. This assumption has not been validated and if untrue may bias
the accuracy of the results in a systematic manner. Bacterial cells contain more than one
complete genome during growth and cell division phases of their life cycle. The number of
genomic copies depends on their growth rate and cell division time. More than one cell division
cycle is often required to complete replication of the genome during rapid log-phase growth and
cell division. The 16S rRNA genes are replicated early in the cell cycle maximizing the number
of 16S rRNA gene copies present in cells during log phase growth. This facilitates the enhanced
ribosome production needed for the high level of protein translation needed during rapid cell
growth and division.
[This section will be updated after validation study]
15.0 Pollution Prevention
15.1 The solutions and reagents used in this method pose little threat to the environment when
recycled and managed properly.
15.2 Solutions and reagents should be prepared in volumes consistent with laboratory use to minimize
the volume of expired materials to be disposed.
16.0 Waste Management
16.1 It is the laboratory's responsibility to comply with all federal, state, and local regulations
governing waste management, particularly the biohazard and hazardous waste identification rules
and land disposal restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Compliance with all sewage
discharge permits and regulations is also required.
16.2 Samples, reference materials, and equipment known or suspected to have viable Bacteroidales
attached or contained must be sterilized prior to disposal.
16.3 For further information on waste management, consult "The Waste Management Manual for
Laboratory Personnel" and "Less Is Better: Laboratory Chemical Management for Waste
Reduction," both available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.
25
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Method B
17.0 References
17.1 Anonymous. 1997. User Bulletin #2. ABI Prism 7700 Sequence Detection System. Foster City,
CA, Applied Biosystems.
17.2 Bordner, R., J.A. Winter, and P.V. Scarpino (eds.). Microbiological Methods for Monitoring the
Environment: Water and Wastes, EPA-600/8-78-017. Cincinnati, OH: U.S. Environmental
Protection Agency, 1978.
17.3 Dick, L.K., and K.G. Field. 2004. Rapid Estimation of Numbers of Fecal Bacteroidetes by use
of a Quantitative PCR Assay for 16S rRNA Genes. Appl. Environ. Microbiol. 70:5695-5697.
17.4 Haugland, R.A., S.C. Siefring, L.J. Wymer, K.P. Brenner, and A. P. Dufour. 2005. Comparison
of Enterococcus Density Measurements by Quantitative Polymerase Chain Reaction and
Membrane Filter Analysis at Two Freshwater Recreational Beaches. Water Research 39:559-
568.
17.5 Siefring, S., M. Varma, E Atikovic, L. Wymer, and R.A. Haugland. 207. Improved Real-Time
PCR Assays for the Detection of Fecal Indicator Bacteria in Surface Waters With Different
Instrument and Reagent Systems. J. Water Health 6:225-237.
17.6 US EPA. 2010. Method A: Enterococci in Water by Taqman® Quantitative Polymerase Chain
Reaction (qPCR) Assay. EPA-821-R-10-004. April 2010.
17.7 Wade, T.J., Calderon, R.L., Sams, E., Beach, M., Brenner, K.P. & Dufour, A.P. 2006. Rapidly
measured indicators of recreational water quality are predictive of swimming associated
gastrointestinal illness. Environmental Health Perspectives 114: 24-28.
18.0 Acronyms
AACT comparative cycle threshold calculation method
AF amplification factor
BAP CDC anaerobe tryptic soy agar with 5% sheep blood
BSA bovine serum albumin
CC cubic centimeter
CMCB chopped meat carbohydrate broth
CPU colony forming units
DNA deoxyribonucleic acid
EDTA ethylenediaminetetraacetic acid
NTC no template control
PBS phosphate buffered saline
PCR polymerase chain reaction
qPCR quantitative polymerase chain reaction
SPC sample processing control
TNTC too numerous to count
UV ultraviolet (light)
26
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Method B
Appendix A:
ABI 7900 Sequence Detector Operation
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Method B
Appendix A - ABI 7900 and ABI 7500 (non-Fast) Sequence Detector
Operation
1.0 ABI 7900 Sequence Detector Operation
1.0.1 Turn on the ABI Model 7900 sequencer and then the computer. Launch the SDS 2.2.2
software program by double clicking on its icon on the computer desktop or from the
Computer Programs menu. The computer will establish communication with the 7900
instrument and if the connection is successful, the software will display the Connected
icon in the status bar when a plate document is opened.
1.0.2 Under File menu, select New.
1.0.3 In resulting New Document window that appears, change container selection from 384
well clear plate to 96 well clear plate using drop down menu. Accept default selections
of Absolute Quantification and Blank Template. Click OK to display a new plate
document.
1.0.4 Click, hold and drag mouse over all PCR reaction tray wells containing samples in upper
left window. Selected wells will be outlined with a bold line and their position numbers
should appear in the results table in the lower left window. To unselect wells, repeat
above process while holding down control key.
1.0.5 Above right hand window, click on Setup tab.
1.0.6 Click on Add Detector button at the bottom of the setup screen.
1.0.7 Click on New in the pop-up window that appears. Another pop-up window will appears.
Under Name, type in a name for the detector that will be used by this method (e.g.,
"Method B"). Under Group select Default. Under Reporter select FAM. Under
Quencher select TAMRA. Click on OK to close second pop-up window. This step only
needs to be performed before the initial analysis run of the method. The detector that is
named is selected in all subsequent analysis runs as indicated in step 1.7)
1.0.8 In pop-up window that was opened in step 1.6, select the desired detector under Names
menu (e.g., Method B) and click on Copy to Plate Document button. Click on Done
button to return to setup screen.
1.0.9 Click on Use box next to FAM detector in right hand window. This box should become
marked with an X. Name and color code for FAM detector should appear in each of the
selected well positions in the upper left window and a data column for this detector should
be created for each of the selected well positions in the results table in the lower left
window.
1.0.10 Click on Instrument tab right hand window.
1.0.11 In instrument screen, change sample volume to 25 uL and choose 9600 emulation.
1.0.12 Still in instrument screen, click on Connect, then click on Open/Close button in lower
right hand "Real Time" window to open PCR reaction tray holder door on instrument.
1.0.13 Insert PCR reaction tray with prepared reactions in holder.
1.0.14 Click on Open/Close button to close PCR reaction tray holder door on instrument.
1.0.15 Click on Start button in lower right hand "Real Time" window to start thermal cycling in
instrument.
1.0.16 Name run file at prompt.
A-l
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Method B
1.0.17 At termination of the run, instrument-calculated cycle threshold values should
automatically appear for each well position and detector entry in the lower left hand
results table window.
1.0.18 At termination of the run success complete, choose Analysis Settings from the toolbar. In
that box enter a value for the Manual Ct Threshold (see 1.18). Click on OK. Click on
Analyze from the toolbar. You should see Ct values in the Results Table.
1.0.19 The instrument-selected threshold line is indicated by the bold red line on the plot and the
value is listed below the window. This can be manually adjusted by either click-hold and
dragging the line up or down or entering new values below. Note: Based on results thus
far with the instrument, the threshold value can be adjusted from the default value of 0.2 to
0.03, however, this should be done only if the threshold line remains above the
background values (seen before the growth curves) for all of the samples.
1.0.20 Calculated CT values for each of the sample tray positions in the lower left hand "Results
Table" will automatically be updated following adjustments of the threshold line. Once
the threshold is adjusted to the desired level, select "Print Report" under the "File" menu.
Check or uncheck desired report items by clicking on their associated boxes and the click
on "Print" button. Note: Minimum report should have both detector boxes, i.e., "FAM"
checked which will show CT values for all selected tray positions for this detector.
1.0.21 Export data by clicking on File from the toolbar. From the drop down menu choose
Export. In the box you will see Look in: and here you choose a directory to send the
exported file too. Click on Export. Save changes to document? will appear, click on
Yes. Click OK.
1.1 ABI 7500 (non-Fast) Sequence Detector Operation
1.1.1 Turn on the ABI Model 7500 sequencer and then the computer. Launch the software
program by double clicking on its icon on the computer desktop or from the Computer
Programs menu. The computer will establish communication with the 7500 instrument.
See "How to Set Up a New Experiment Using the ABI 7500" for screen shots. (Note: All
screen shots were taken for Method A, a qPCR method for Enterococcus. Red
instructional text in boxes in the screen shots has been changed for Method B. Please
carefully read the text here to verify the correct parameters for this method.)
1.1.2 Click on either the New Experiment or Advanced Setup button to create a new
experiment, which will pull up the Experiment Menu.
1.1.3 From the Setup menu, select Experimental Properties to select the experiment type and
give the experiment a name. Enter the experiment name in the asterisked box.
1.1.3.1 Click on 7500 (96 Wells) to select the instrument type. Note: This protocol is
not designed for the 7500 Fast.
1.1.3.2 Scroll down to access more experiment properties options. Click on
Quantitation - Standard Curve to select the experiment type
1.1.3.3 Click on TaqMan® Reagents to select the reagents used.
1.1.4 From the Setup menu, select Plate Setup. Click on the Define Targets and Samples tab
to define the reporter-quencher dye for each target and also to enter the sample
identifications (e.g., Bact 5X).
1.1.4.1 Click on Add New Target and enter the name of the target. Under the
Reporter heading, click on the drop down menu to select the reporter dye
A-2
-------
Method B
FAM. Under the Quencher heading, click on the drop down menu to select
TAMRA as the quencher.
1.1.4.2 Repeat the process in Section 1.1.4.1 to add more than one target, e.g., Sketa
5x.
1.1.4.3 In the Define Samples section, click on Add New Sample and enter the
sample name in the Sample 1 box.
1.1.4.4 Click on the Assign Targets and Sample tab to assign the Target, sample ID
and sample type to the wells.
1.1.4.5 Drag the mouse over the desired cells to assign the Target, Task
and Sample.
1.1.4.6 While the cells are highlighted, in the Assign target(s) to the
selected wells section, check the box under Assign for the
appropriate Target, then under Task, select U for unknown, S for
standard or N for negative control.
1.1.4.7 In the Assign sample(s) to the selected wells section, check the
box under Assign to label the highlighted cells as Sample 1 (e.g.).
1.1.4.8 Repeat the process in Section 1.1.4.4, dragging the mouse over the appropriate
cells, and then assigning them as Unknowns, Standards or Negative Controls,
as appropriate. Note that for the Standards, in the Assign target(s) to the
selected wells section, if there are 4 different values, each cell will need to be
highlighted separately, and a quantity (e.g. 40000.0) typed in the Quantity
box.
1.1.4.9 Click on Print Report at the top of the screen to print a plate layout for
loading master mix and sample extracts.
1.1.4.9.1 Select Plate Layout option by clicking on the box next to it.
1.1.4.9.2 Click Print Report to print the plate layout.
1.1.5 From the Setup menu, select Run Method to set up the thermo cycling profile.
1.1.5.1 In the Graphical View tab, change the Reaction Volume Per Well from the
default 50 uL to 25 uL by typing in 25.
1.1.5.2 Under Cycling Stage section, change the Number of Cycles to 45.
1.1.5.3 Check that the default settings of Temperature and Time for the two Holding
Stages and the Cycling Stage are correct. Specifically, these should be:
Holding Stage 1: 50.0°C for 2:00 minutes, Holding Stage 2: 95.0°C for 10:00
minutes, and Cycling Stage: 95.0°C for 0:15 seconds. (Note: The purpose of
Holding Stage 1 is to eliminate potential PCR carryover products in the
reactions using the Amp-Erase® UNG enzyme provided in the TaqMan®
Universal PCR Master Mix. The purpose of Holding Stage 2 is to inactivate
the Amp-Erase® UNG enzyme.)
1.1.5.4 The second step of the Cycling Stage is defaulted at 60.0°C for 1 minute -
change the time to 2:00 minutes. (Note: This sets the detection of the
fluorescence signal to occur at the end of the second step in each cycle.)
1.1.5.5 Click on Save at the top of the screen to save the run before actually running
the experiment.
A-l
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Method B
1.1.5.6 The Save screen will open so that you may select the folder to save the Run
Data File. Type in the Experiment Name (if you did not already do so at the
beginning of the Experiment Setup) and click on Save. If you had previously
typed in an Experiment Name and there is no change, click on Save. (Note:
You must save to the hard drive (rather than a flash drive).)
1.1.6 From the Experiment menu, select Run to monitor the run in real time.
1.1.6.1 The Run Status screen will open - click on Start Run. When the run has
successfully started, the Start Run button will turn change from green to red.
1.1.7 When the run has completed, from the Setup menu, select Analysis to export your
results. See "How to Export Results from the ABI 7500 Software" for screen shots.
1.1.7.1 The Analysis Settings for Experiment name screen will open.
1.1.7.2 Under the CT Settings tab, click on the Target desired to be set from Default
to Manual Threshold setting (generally, this would be the Bact).
1.1.7.3 In the CT Settings for Bact section, uncheck the boxes next to Use Default
Settings, and Automatic Threshold. Enter the desired Threshold (e.g.,
0.025). If more than one target is present, highlight the next target (e.g.,
Sketa) and repeat this step.
1.1.7.4 Click on Apply Analysis Settings to save the changes.
1.1.8 Click on Export at the top of the screen to save the experiment results data and to save the
selected plots required in the report. Note that the threshold value will have changed on
the Amplification plot curve graphic.
1.1.8.1 The Export Data screen will open. In the Select data to export section, check
all of the boxes (i.e. Sample Setup, Raw Data, Amplification Data, Results,
and Multicomponent Data).
1.1.8.2 For Select one file or separate files, select One file to export all the data into
one file with multiple tabs.
1.1.8.3 For Export File Name, enter the name of the Experimental data file to be
exported, and for File Type, choose *.xls.
1.1.8.4 For Export File Location, click on Browse to select the folder in which you
wish to export your file. Always save your file to the hard drive.
1.1.8.5 Click on Start Export. When this is completed, an Export Completed screen
will open - click on Close Export Tool to complete this task.
1.1.9 How to interpret your results. See "How to Interpret Results from the ABI 7500" for
screen shots.
1.1.9.1 After exporting your data, from the Experiment menu, select Analysis.
1.1.9.1.1 From the Analysis menu, select Amplification Plot to view the
results of the selected samples at different plot settings.
1.1.9.1.2 Drag the mouse over the desired cells to select the samples to be
plotted in the amplification plot.
1.1.9.1.3 In the Amplification Plot section, in the Plot Settings tab, select
the Plot Type (from the drop down menu) and the Graph Type
(from the drop down menu).
A-2
-------
Method B
1.1.9.1.4 If the plot requires the display of the Threshold and Baseline, in
the Options tab, check the boxes next to Threshold and
Baseline. If you want to see the results in tabular format, click on
View Well Table, and scroll down or sideways for more samples
and analysis parameters.
1.1.9.1.5 The toolbar above the graph can be used to zoom in and out,
print, save the file as *.jpg, and change the plot properties.
1.1.9.2 For the Standard Curve, select Standard Curve from the Analysis menu to
view the results of the same sample (assuming they have the same target) and
standards in the plot.
1.1.9.2.1 In the Plot Settings tab, click the drop down menu arrow beside
Target to select a different target (if the standards were analyzed
in more than one target), and click the drop down menu arrow
beside Plot Color to change the plot color.
1.1.9.2.2 The standard curve parameter display below the plot (i.e. Target,
Slope, Y-Inter(cept), R2, and Eff% (percent efficiency) is useful
for data analysis.
1.1.9.3 For Multicomponent plots, click on Multicomponent Plot from the Analysis
menu to view the variation in the fluorescence of the dyes used in the
experiment over the ascending repeat of the 45 thermo cycles based on well,
target, or dye.
1.1.9.3.1 Click on the drop down menu arrow next to Plot Color in the
Plot Settings tab to select the parameter for the basis of the graph
(e.g., well, target, or dye).
1.1.9.4 For raw data plots, select Raw Data Plot from the Analysis menu to view the
variation in the emission of fluorescence of the dyes in different filters present
over the ascending repeat of the 45 thermo cycles.
1.1.9.4.1 In the Options tab, drag the pointer on the cycle scale to the
desired cycle number to view the fluorescence variation through
filters of the samples at that cycle.
1.1.9.5 For quality controls, click on QC Summary from the Analysis menu to view
the analysis summary of the samples.
1.1.9.6 To view all the plots, click on Multiple Plots View from the Analysis menu
to see all of the plots for the samples in a single window. Each plot will have
its own drop down menu as in Sections 1.1.9.1.5..
1.1.10 To print the report, click on Print Report at the top of the screen.
1.1.10.1 Check all of the boxes (/'. e., Experimental Summary, Results Summary, Plate
Layout, Amplification Plot (3 boxes), Standard Curves, Results Table, and
QC Summary).
1.1.10.2 Click on Print Report at the bottom of the screen
A-l
-------
How to Set Up a New Experiment Using the ABI 7500
7500 Software v2.0.1
File Edit instrument Analysis Tools Help
[iiiTI New Experiment fr L_> Open... I ^}
Analyze Experiment
Click either button to
create new experiment
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Experiment Menu«
Setup
Experiment Properties
:!: Plate Setup
Run Method
Reaction Setup
.erials List
Run
Analysis
Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
Experiment Properties
iteran experiment name, select the i
PCR reactions and instrument run.
1. Click "Experiment Properties" to
select the experiment type and give
an experiment name
How do you want to identify this experiment'!
' Experiment Name: ' Untitled
Barcode (Optional):
User Name (Optional)^
Comments (Optic
WhLh instrument are you using to run the experiment?
7500 (96 Wells)
7500 Fast (96 Wells)
Set up, run, and analyze an experiment using a 4- or 5-color, 96-well system.
2. Enter the
at type of experiment do you want to set up?
Experiment Name f7
Quantitation - Standard Curve
Quantitation - Relative standard Curve
Quantitation - Comparative CT (AACi
Melt Curve
Genotyping
Presence/Absence
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New Experiment - t3 Open... A Save ' ^ close ^ Export... - ^ Print Report...
Experiment Menu«
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Experiment Properties
Plate Setup
Run Method
Reaction Setup
.erials List
Run
Analysis
START RUN
Expe... NRSA Batch! ENT 03 ... Standard GUI Reag... TaqMan® Reagen
Experiment Properties
Enter an experiment name, select the instrument type, select the type of experiment to setup, then select materials and methods fort
PCR reactions and instrument run.
How do you want to identify this experiment!
' Experiment Name:
Barcode (Optional):
User Name (Optional):
Comments (Optional):
NRSA Batch 1 ENT 032310|
Which instrument are you using to run the experiment?
/ 7500 (96 Wells)
7500 Fast (96 Wells)
p, run, and analyze an experiment using a 4- or 5-color, 96-well system.
/ Quantitation - Standard Curve
Click the type of instrument
used - in this case it is the
7500 (96 wells) plate
Quantitation - Relative standard Curve Quantitation - Comparative CT (AACr
Scroll down for more
experiment properties
options
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Experiment Menu«
Setup
Experiment Properties
Plate Setup
Run Method
Reaction Setup
.erials List
Run
Analysis
Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
Experiment Properties
Which instrument are you using to run the experiment?
START RUN ft
7500 (96 Wells)
7500 Fast (96 Wells)
Set up, run, and analyze an experiment using a 4- or 5-color, 96-well system.
What type of experiment do you want to set up?
Quantitation - Standard Curve
Quantitation - Relative Standard Curve Quantitation - Comparative CT (AACi
vie It Curve
Genotyping
Presence/Absence
Use standards to determine the absolute quantity of target nucleic acid sequence in samples.
Which reagents do you want to use to detect the target sequence:
TaqMan® Reagents
Green Reagents
Other
The PCR reactions contain primers designed to amplify the target sequence and a TaqMan® probe designed to detect amplification of th
target sequence.
Click to select
experiment type
liich ramp speed do you want to use in the instrument run':
[standard (~ 2 hours to complete a run)
-
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New Experiment - t3 Open... A Save ' ^ close ^ Export... - ^ Print Report...
Experiment Menu«
Setup
Experiment Properties
Plate Setup
Run Method
Reaction Setup
.erials List
Run
Analysis
Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
Experiment Properties
Which instrument are you using to run the experiment?
START RUN ft
7500 (96 Wells)
7500 Fast (96 Wells)
Set up, run, and analyze an experiment using a 4- or 5-color, 96-well system.
What type of experiment do you want to set up?
Ouantitation - Standard Curve
Quantitation - Relative Standard Curve Quantitation - Comparative CT (AACi
vie It Curve
Genotyping
Presence/Absence
Use standards to determine the absolute quantity of target nucleic acid sequence in samples.
Which reagents do you want to use to detect the target sequence:
TaqMan® Reagents
Green Reagents
Other
The PCR reactions contain primers designed to amplify the target sequence and a TaqMan® probe designed to detect amplification of th
target sequence.
Click on TaqMan® Reagents to
select the reagents used
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7500 Software v2.0.1
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New Experiment - :_j Open...
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Experiment Menu«
U Setup
Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
START RUN »
1. Click on
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Define Targets and Samples Assign Targets and Samples
^^_ I
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Instructions: Define trrejargets to quantify and the samples to test in the reaction plate.
Define Tarnete
Define Qamnlec
2. Click "Define Targets and Samples" tab to define
the reporter-quencher dye for each target and also
enter sample IDs.
L
Define Biological Replicate Groups
|JP Instructions: For each biological replicate group in the reaction plate, click Add Biological Group, then define the biological group.
Add Biological Group I Delete Biological <
Biological Group Name
Color
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Assign Targets and Samples
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Experiment Menu«
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'tit; I Plate Setup
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.erials List
Run
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2. Enter the
Target Name
Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
START RUN
Define Targets and Samples
Q Instructions: Define the targets to quar
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f) Instructions: For each /ological replicate
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Add Biological Group JOelete Biological Grant
3. Select the Reporter
dye FAM from the
drop down menu
4. Select the Quencher
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down menu
Assign Targets and Samples
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.erials List
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Expe... NRSA Batch! ENT 03 ... Standard GUI Reag... TaqMan® Reagen
START RUN
Define Targets and Samples | Assign Targets and Samples
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New Experiment - t3 Open..
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Experiment Menu«
Setup
Experiment Properties
'tit; I Plate Setup
Run Method
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.erials List
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START RUN
Define Targets and Samples ] Assign Targets and Samples
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Setup
Experiment Properties
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Experiment: Untitled Type: Standard Curve Reagents: TaqMan® Reagents
START RUN
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"^^^^»^^^^^^^^^^^^™^^^^^^^^^^^^^^^^^J=
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Experime... NRSA Batch 1 ENT 03231C Ty... Star
Define Targets and Samples T Assign Targejg am
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Instructions: jo set up unknowns: Select^H^'assign targej^ se
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n^ Define and Set Up Standards
While the cells are highlighted,
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View Plate Layout I View well I able
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New Experiment - t3 Open...
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Experi
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To designate standards,
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jrve Reagen... TaqMan® Reagents
START RUN ft
es
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lecf'N" (Negative Control) as the task for each target assignment.
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Type in a Quantity for each
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different values, highlight
each cell independently and
fill in the quantity
Print Report...
Standard Curve Reagen... TaqMan® Reagents
START RUN ft
s and Samples
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Ex... NRSA2009 Batch34 ENT C ... StanoS^ Cu Rea... TaqMan® Reager
START RUN ft
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To set uc
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ment, then
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Q Select data for the report. Click "Pr
D Experiment Summary
~H Results Summary
Run
Analysis
1. Select "Plate Layout" option by
clicking in the box next to it
late
dev), CT (mean) and CT (std dev).
An illustration of the wells in the reaction plate. Displays the contents assigned to each well.
D Amplification Plot (ARn vs. Cycle) Data collected during the cycling or amplification stage. Displays baseline-corrected
normalized reporter (ARn) plotted against cycle number.
D Amplification Plot (Rn vs. Cycle) Data collected during the cycling or amplification stage. Displays normalized reporter (Rn)
plotted against cycle number.
D Amplification Plot (Civs. Well) Data collected during the cycling or amplification stage. Displays CT plotted against well
number.
The best fit line using CT values from the standard reactions plotted against standard
quantities.
A table of experiment results for each well, including sample, target, task, quantity, ARn and CT.
D Standard Curves
D Results Table (By Well)
D QC Summary
A table of flags applied to wells in the experirc
occurrence,and a list of flagged wells.
Print Preview
2. Click "Print Report"
to print the plate
layout.
ignment, then
for each target
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Save '
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Export... - g Print Report...
Experiment Menu«
Setup
Experiment Properties
Plate Setup
Run Method
Reaction Setup
Materials List
: Run
Analysis
Expe... NRSA Batch! ENT 03 ... Standard GUI Reag... TaqMan® Reagen
START RUN ft
Select "Run Method" to set
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md Samples
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select "U" (Unknown) as the task for each target assignment, then
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Experime... NRSA Batch! ENT03231C Ty... Standard Curve Reagen... Taq Man® Reagents
START RUN
Q Review the reaction volume and the thermal profil
Graphical View ]
Reaction Volume Per Well
1. Change the Reaction Volume Per
Well from the default of 50uL to 25uL
Expert Mode
from the library.
Add Stage T Add Step T Delete Selected Undo "Set Hold Time"
(nothing to
Collect Data T
Open Run Method
Holding Stage
Holding Stage
2. Change the Number of Cycles to 45
Cycling Stage
Number of Cycles: |45
I i tname Auioueiia
Starting Cycle:
3. Confirm that the default Temperature
and Time settings in both Holding
Stages and in step 1 of the Cycling
Stage are as displayed - if not, reset
them
v
Delta Off
4. In the data collection step,
change the time frame from
the default 1 minute to 2
minutes
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Experiment Propertie
Plate Setup
Rim Method
Reaction S
Materials Li
: Run
Analysis
nfilled x
1. Click to save the run
before starting the run
sagen
START RUN
Save inl
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Comparative Ct Example.eds
Genotyping Example.eds
Presence Absence Example.eds
Relative Standard Curve Example.eds
1 Standard Curve Example.eds
\
2. Select the folder for
saving the Run Data file
File name:
NRSA Batch 1 ENT 032310.eds
Files of type: Experiment Document Single files (*eds)
£
- Legend
ethod or select a run
60.0°C
02:00
Dal
3. Enter the experiment name if not entered at the
beginning of the experiment setup and click "Save"
or if already have the appropriate experiment name,
without making any changes click "Save"
ted
>J «'*> *
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
New Experiment - i_3 Open... d Save" Jj close 4& Export... - Q Print Report...
Experiment Mem
Click "Run" to monitor the run in real-time
Setup
Run
Amplification Plot
Temperature Plot
Run Method
rfication Settings
ntitled x
s: TaqMan® Reagents
Instrument Status: . Disconnected
Click to start the run. The
green box will turn red when
the run has been started
successfully
1D
1
0.1
D.D1 •
0.001
0.0001
0.00001
0.000001
Amplification Plot
2 i 6 8 10 12 14 18 18 20 22 2i 23 28 30 32 3* 38 JB 40
Cycle
- Lcytsiul
B A B " C ~ D
IE BF BO BH
Enable Notifications
Disconnected
TDF 1806
-------
How to Export Results from the ABI 7500 Software
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
[UiT| New Experiment - i_J Open... i«j Save - LJ" Close L^> Export... - ^ Print Report...
Experiment Menu«
Setup
Run
' Analysis
Amplification Plot
Standard Curve
Mutticomponeiit Plot
Raw Data Plot
QC Summary
Multiple Plots View
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reagt
Amplification Plot
< View Plate Layout T View Well Table
^ Plot Settings
Click on the "Analysis" tab in the Experiment Menu
to access the "Analysis Settings"
10
1
0.1
0.01
0.001 •
0.0001
0.00001
0.000001
0.0000001
Amplification Plot
t 5 3 10 12 14 16 13 20 22 3* 23
Cycle
A B C D E •F •G •H
fOptions\
Target:
Threshold:
1 2 3 4 5 6 7 8 9 10 11 12
2 ^ m..' ci.' rs
m c 90... 100 Jr
69..
Wells: [a|40 Unknown \~\ 16 Standard [~] 0 Negative Control
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 1 4 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds
Disconnected
start
baya... •' Verb.
,* m 0 '
-------
7500 Software v2.0.1
File Edit Instrumei
MI
[siT| New Experiment -
Experiment
Setup
Run
Analysis
.plrficalion P
Standard Curvn
Multicomponei
Raw Data Plot
OC Summary
Multiple Plots \
1. To change the threshold setting from
Automatic to Manual, click on "Analysis
Settings"
-i
Analysis Settings for NRSA Batch 34 test run 022310
CT Settings T Flag Settings Advanced Settings
Q Review the default settings for
different settings for a target, s
Default CT Settings
Default CT settings are used to calc
Threshold: AUTO Baseline St
2. Under the "CT Settings" tab,
click on the Target desired to
be set from Default to Manual
Threshold setting
it Default Settings." To use
'i the settings that are display
.click "Edit Default Settings."
Select a Target
Tarnst Threshnld
ENT
SKETA
AUTO
AUTO
Haseline Start
AUTO
AUTO
Hasplinp Fnrl
AUTO
AUTO
3. Unc
"1
CT Settings for ENJ_
CT Settings to Use:
ftutamaticjrtreshold
Hires hoU/l '.< .
Defau|t Settjngsi
;/j Automatic Baseline
Sseline Start Cycle: I TT] End Cycle I 15
3. Uncheck the box next to
"Use Default Settings"
Revert to Default Analysis Settings
Apply Analysis Settings
12
OD
Cancel I Control
Analysis Summary Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 14 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gTj New Experiment - t3 Open...
Save '
close
Export... - ^ Print Report...
Experiment Menu«
Setup
Run
Analysis
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reage
Analysis Settings for NRSA Batch 34 test run 022310
•plification P
Standard Curvn
Multicomponei
Raw Data Plot
OC Summary
Multiple Plots \
CT Settings T Flag Settings Advanced Settings
Q Reviewthe default settings for analysis oftargets in this experiment. To editthe default settings, click"Edit Default Settings." To use
different settings for a target, select the target from the table, deselect "Use Default Settings," then change the settings that are display
Default CT Settings
Defaulter settings are used to calculate the CT for targets with out custom settings. To editthe default settings, click"Edit Default Settings."
Threshold: AUTO Baseline Start Cycle: AUTO Baseline End Cycle: AUTO | Edit Default Settings |
- Select a Target -
Target Threshold
ENT
AUTO
SKE
Baseline Start
AUTO
Uncheck the box next to
"Automatic Threshold" to
enter the desired threshold
CT Settings for ENT
CT SettingstoUse: D Pse^Defau|t Settjng^
lutomatic Threshold
Threshold:
0 Automatic Baseline
Baseline Start Cycle: I 3 $1 En.l C /cle I 15
Revert to Default Analysis Settings
Apply Analysis Settings
12
OD
Cancel I Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 14 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gTj New Experiment - t3 Open...
Save '
close
Export... - ^ Print Report...
Experiment Menu«
TanMan®
R
Analyze
Analysis Settin
• Analysis Settings for NRSA Batch 34 testrun 022310
Home
CT Settings T Rag Settings T Advanced Settings
Q Reviewthe default settings for analysis oftargets in this experiment. To editthe default settings, click "Edit Default Settings." To use different
settings for a target, select the target from the table, deselect "Use Default Settings," then change the settings that are displayed.
Default CT Settings
Defaulter settings are used to calculate the Crfortargets without custom settings. To editthe default settings, click "Edit Default Settings."
Threshold: AUTO Baseline Start Cycle: AUTO Baseline End Cycle: AUTO | Edit Default Settings
Select a Target
Target
ENT
SKETA
Threshold
0.2
0.2
Baseline Start
AUTO
AUTO
Baseline End
AUTO
AUTO
A
CT Settings for the 2 Selected Targets
CT Settings to Use: Q Use Default Settings
H Automatic Threshold
Enter the desired Threshold.
Threshold: 0.025
0 Automatic Baseline
Baseline y. ' 3 I iriv! , l^ I 15
*lf more than one target is present, highlight the next
target and repeat this process
Revert to Default Analysis Settings
Apply Analysis Settings
Cancel
-
B
12
ENT
• I In
I -
ENT
-• I In
I .
ENT
• I In
I -
ENT
• I In
|>
ntrol
Analysis Summary Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 40 Wells Omitted by Ana... 0 Samples...8 Targets...1
NRSA Batch 34...032310 eds.eds x
Disconnected
%' 7500 S,
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT] New Experiment - u3 Open... A Save ' ^ Close ^Export...- ^ Print Report...
Experiment Menu«
Setup
Run
Analysis
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reage
Analysis Settings for NRSA Batch 34 testrun 022310
pimcation P
Multicomponei
Raw Data Plot
QC Summary
Multiple Plots \
CT Settings T Flag Settings Advanced Settings
Q Reviewthe default settings for analysis oftargets in this experiment. To editthe default settings, click"Edit Default Settings." To use
different settings for a target, select the target from the table, deselect "Use Default Settings," then change the settings that are display
Default CT Settings
Default CT settings are used to calculate the CT for targets with out custom settings. To editthe default settings, click"Edit Default Settings."
Threshold: AUTO Baseline Start Cycle: AUTO Baseline End Cycle: AUTO \ Edit Default Settings |
- Select a Target
Target
ENT
SKETA
LI
CT Settings for SKETA
Select "Apply Analysis Settings" to save
the changes
Jse Default Settings
0 Automatic Baseline
Baseline Start Cycle | 3 | Eivl '1 yule | 1 :
Revert to Default Analysis Settings
Apply Analysis Settings
Cancel
1
12
OD
i m
T- r-i-
i-
Ir
H ^
]
ontrol
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 14 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
^Home H NRSA Batch 34...032310eds.eds x
Disconnected
Start ® * f:
7500,., 04M,
10:40 AM
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
New Experiment - i_3 Open... aJ Save ~ LJI' Close
Export..
j Print Report...
Experiment Menu«
Setup
Run
Analysis
•plification Plot
Standard Curve
Multicomponent Plot
Raw Data Plot
OC Summary
y-fr J -I- j^_ j~>4
i-j-^ ^^ j-i j-yg- j-
Click on "Export" to save experiment results
data and to save the selected plots required
in the report
v
Amplification Plot
Show in Wei...
View Well Table
ct Item -
-SelectItem- v
ra View Legend
B
9 10 11 12
ENT
2 ^ ^SEN?,
i H.IVM pj. inn
1 ENT ' uu TIO
3 ENT r1 -T.. 228....
". i ,n 98.19 CT-1- C.T 1
1121
0 ENT 0 ENT [H ENT £3 ENT
23.78 63.45 400 34.99
Note that the threshold value has changed on the Amplification Plot
El ENT 4E4 ^^^ s £jj ENT
12.63 CT:... ~r' 4528
1 ^^EN? 1 ^ 1 T
[Tj ENT 4E4 j ENT ! J ENT
265.... 47.65
Options
Show: 0 Threshold — D Baseline Start: Well Target ^
EN?1
304.2 !^3 ^ENT
CT: ... T>J
Wells: Ly 40 Unknown M 16 Standard [Tjo Negative Control
Analysis Summary Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 35 Wells Omitted by Ana... 0 Samples...8 Targets...1
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
."• I 11:16 AM
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
-i
~j^\ New Experiment - i_3 Open... jj Save » _j Close
Export...
4) Print Report...
Experimen
Export Data
Check the boxes next
to the desired results to
Setup
Run
Analysis
Q Select the type of data to export, select whetherto export one file or separatefifesj OXDOft
Click"Customize Export" to change the export format and to select fields tc^cpori H
Export Properties Customize Export
1. Select data to export:
0 Results
0 Raw Data 0 MulticomponentData
0 Amplification Data
2. S e I e ct o n e fi I e o r s e p a rate fi I e s: OneFile v Select to export ati data in one file or in separate files for each data t/t
3. Enter export file properties:
Export File Name:
Export File Location:
NRSA Batch 34 testrun 02231 0_data
File Type: 2S] (*.xls) v
C:\Applied Biosystems\75QQ\experiments
Browse
D Open file(s) when export is complete
^j Save current settings as the default
Start Export | | Cancel
ectltem- v
10 11 12
RI ra r--r
4E4 "
4t4 ^T.
1 [-«, bJ •
.. 4E3 r,T.
anl
4E3 JC
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1 El.
m n-r-
^'-*
1 BI.
egative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT) New Experiment - i_3 Open... ^J Save- Jj close
Export...
4) Print Report...
Experimen
Export Data
Setup
Run
Analysis
Q Select the type of data to export, select whether to export one file or separate files, then enter export file properties. (Optional)
Click "Customize Export" to change the export format and to selectfields to export. Click"Start Export" to export your data.
ble
Export Properties Customize Export
1. Select data to export:
0 Sample Setup 0 Results,
0 Raw Data M
0 Amplification Data
From the drop-down menu select
"One File" to export all data into a
single file with multiple tabs
2. Select one file or separate files
3. Enter export file properties:
Export File Name:
Export File Location:
Select to export alt data in one file or in separate files for each data t/t
NRSA Batch 34 testrun 022310 data
File Type:
C:\Applied Biosystems\75QQ\experiments
Browse
D Open file(s) when export is complete
^j Save current settings as the default
Start Export | | Cancel
^ El-1 •} ^m l
c 10.. m r.T."
ETI ra
4E4 H"
I —I r-.-j v
i n i • ' T
4E4 r!."
4E3 C
^"
1 El
m n-r-
^I:A
1 13.
Jegative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT) New Expert me nt - i_3 Open... ^J Save- Jj close
Export...
4) Print Report...
Experimen
Setup
Run
Analysis
Q Select the type of data to export, select whether to export one file or separate files, then enter export file properties. (Optional)
Click"Customize Export" to change the export format and to selectfields to export. Click"Start Export" to export your data.
Export Properties Customize Export
1. Select data to export:
0 Sample Setup
0 Raw Data
0 Amplification Data
0 Results
0 MulticomponentData
2. Select one file or separate files:
3. Enter export file properties:
One File
Select
Enter the name of the
experiment data file to be
eic^orfec
Export File Name: I
Export File Location:
ISA Batch 34 testrun 032310 data
iFileType:
C:\AppliedBiosystems\75QQ\experiments
Browse
D Open file(s) when export is complete
^j Save current settings as the default
Start Export | | Cancel
Die
ectltem- v
) 10 11 12
1 1
Si' so.,
4E4 ^T.
m~l™1
u- [3
i 4E4 ^1
.. 4E3 Jtt"
1 E3-.
1 BI.
Megative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT) New Experiment - i_3 Open... ^J Save- Jj close
Export...
4) Print Report...
Experimen
Setup
Run
Analysis
Select the type of data to export, select whetherto export one file or separate files, then enter export file properties. (Optional)
Click "Customize Export" to change the export format and to select fields to export. Click"Start Export" to export your data.
Export Properties Customize Export
1. Select data to export:
0 Sample Setup
0 Raw Data
0 Amplification Data
0 Results
0 MulticomponentData
2. Select c
3. Entere)
Select *.xls (Excel) from the "File Type"
drop-down menu
ife files for each data t/t
Export File Name:
Export File Location:
NRSA Batch 34 testrun 03231 0_data
C:\Applied Biosystems\75QQ\experiments
^V^ileT^
e:
(*xls)
HJ r.
D Open file(s) when export is complete
^j Save current settings as the default
Start Export | | Cancel
Die
ectltem- v
) 10 11 12
c 10.. rn
^ ^
1 1
Si' so.,
4E4 ^T.
m~l™1
u- [3
i 4E4 ^1
i.. 4E3 "."
I 4E3 S."
1 B-.
1 BI.
Megative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT) New Expert me nt - i_3 Open... ^J Save- Jj close
Export...
4) Print Report...
Experimen
Setup
Run
Analysis
Select the type of data to export, select whetherto export one file or separate files, then enter export file properties. (Optional)
Click "Customize Export" to change the export format and to select fields to export. Click"Start Export" to export your data.
Export Properties Customize Export
1. Select data to export:
0 !?.a.m.PJ.?...s.Mu.Pi
0 Raw Data
0 Amplification Data
0 Results
0 MulticomponentData
2. Select one file or separate files: One File
3. Enter export file properties:
v Select to export ail data in one file or in separate files for each data tyi
Export File Name:
Export File Location:
NRSA Batch 34 testrun 022310 data
File Type: SSj (*xls) v
C:\Applied Biosystems\75QQ\experiments
Browse
D Open file(s) when export is complete
Click on "Browse" to select the
folder / location to export the File
^j Save current settings as the default
Start Export | | Cancel
Die
ectltem- v
) 10 11 12
= 1D-. m
1 1
Si' so.,
4E4 ^T.
4E4 "."
i.. 4E3 ^~."
4E3 S."
1 IB..
1 ^'S^
Megative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
gT) New Experiment - i_3 Open... ^J Save- Jj close
Export...
4) Print Report...
Experimen
Setup
Run
Analysis
Select the type of data to export, select whetherto export one file or separate files, then enter export file properties. (Optional)
Click "Customize Export" to change the export format and to select fields to export. Click"Start Export" to export your data.
Export Properties Customize Export
1. Select data to export:
0 Sample Setup
0 Raw Data
0 Amplification Data
0 Results
0 MulticomponentData
2. Select one file or separate files: One File
3. Enter export file properties:
v Select to export ail data in one file or in separate files for each data t/t
Export File Name:
Export File Location:
NRSA Batch 34 testrun 03231 0_data
File Type:
® rxls) v-
C:\Applied Biosystems\7500\experiments
Browse
D Open file(s) when export is complete
Click on "Start Export"
to export the data to the
determined location
^j Save current settings as the default
Die
ectltem- v
) 10 11 12
1 1
Si' so.,
4E4 ^T.
i 4E4
F~| ~"| f-iH ^
.. LJ.. m
.. 4E3 "."
I 4E3 JC"
1 B-.
i m f>-r-
1 El.
r~l r-r-
Megative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
[siTI New Experiment - i_3 Open...
Save ~
Close
Experimen
Export...
4) Print Report...
Setup
Run
Q Select the type of data to export, select whetherto export one file or separate files, then enter export file properties. (Optional)
Click"Customize Export" to change the export format and to selectfields to export. Click"Start Export' to export your data.
Analysis
.plrfic jtion F
Standard Cmv
Multicomponei
Raw Data Plot
OC Summary
Multiple Plots \
Expor
A pop-up window "Export Completed" appears
once the exporting of the file is completed.
1. Select data to export:
0 MulticomponentData
2. Select one file o
3. Enter export file
Export File Name:
Export File Locatioi
Export Completed
> ff/es for eacA ctefa f/i
Your file has been exported to ;
C:\Applied Biosystems\75QO\experiments\NRSA Batch 34 testrun 032310_data.xls
What do you want to do next?
Export More Data
Type:
i] (*.xls) v
Browse
D Open file(s) when export is complete
Click on "Close Export Tool" to
close the export window once
data file exporting is completed.
^j Save current settings as the default
Start Export
Cancel
Die
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i ETI n f^-r
'S?i-fl
1 4E4 r^." r^.
5.. 4E3 Jtt"
I 4E3 S."
1 B-.
i m n-r-
1 ^'m^
Megative Control
Analysis Summary Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 24 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home H NRSA Batch 34...032310 eds.eds x
Disconnected
-------
How to Interpret Results From the ABI 7500
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
03 New Experiment - uJ Open... iJ Save •> _j Close L^ Export... - ^ Print Report...
Experiment Menu«
Setup
Run
Analysis
pllfication Plot
Standard Curve
utticomponent Plot
Raw Data Plot
QC Summary
Multiple Plots View
NRS/
Select "Amplification Plot" to view the results of
the selected samples at different plot settings
Settings \_
Plot Type: JARn vs Cycle v| Graph Type: Log v| Plot Co
2 i a a ioisuieiaaaaaaaflaiao32M3Bffl«4344
Cycle
select wens witn: |- aeieci item- >M |- yeieet
:em- v
Show in Wei...
g5@| View Legend
1 2 3 4 5 6 7 8 9 10 11 12
11.. 100 ".
73... 100
c 98... 100
u j-,-. ,a c ao... i uu f-,-.
TV ,^.^
Use these icons to zoom
in/out, print, save the file as
*.jpg, and change the plot
properties
Options
Show: 0 Threshold — Q Baseline Start: Well Target^h. v
32.. ra S" 34.. m S"
^^ "V t-n "^M ~~t ^H r-ji 'V
ffl^ 1 lr EI -1! ^ m^
75... ™ fc! 71... m ^i
71.
Wells: 0]40 Unknown Q 16 Standard Qo Negative Control
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted Man... 0 Wells Fla...1 3 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
Home B MRS A Batch 34...032310 eds.eds x
Disconnected
Google ... fir Pow
-------
7'inn Snftwam v9 n 1
2. Select the
"Plot Type"
from the drop-
down menu
Run
r Analysis
Amplification Plot
Standard Curve
Multicomponent Plot
Raw Data Plot
JC Summary
Multiple Plots View
Analysis Menu-Amplification Plot
I Tools HP!D
Save - LJ3 Close
Export... -
Pt
NRSA Batch 34 testrur ...Star
3. From the "Graph Type"
drop down menu select either
Log or Linear
ettings
Amplification Plot
10 r
Amplification Plot
2 4 e a 10 12 U 1>3 1tl Xl 22 M 26 33 JO 32 Ji -36 38 « *2
IA B CBDBEBFBGBH
. j i aiymi •vi irutjsnuiu. | |«mu
Show: [^Threshold — l~~l Baseline Start: Well Taraet^t^ ^
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted I
Home B NRSA Batch 34...032310 eds.eds x
start m * r:
View Plate Layout | View Well Table
Select Wells With: - Select Item - v
Show in Wei...
lH View Leg end
1
J ^ pn ~ CAL-..^
L&S «*•• j?r «">
£.,J 110.1ECT1... f;r1
1 1
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28.2 73.25 Cr:1... 35.02
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^ t^
22.31
CAL-..^
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357.9
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: 5... 244.5£ 34.58
1. Drag the mouse over the
desired cells to select the
samples to be plotted in the
amplification plot
-------
Analysis Menu-Amplification Plot
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
[siT| New Experiment - \Jj Op
Experiment Menu
2. Select "View Well Table" to view
the results in a tabular format
Setup
'.*»*,
Run
" Analysis
Amplification Plot
Amplification Plot
r Plot Settings \
Plot Type: !ARnvs Cycle V Graph Type: Log vl Plot Co -
-
1. If the plot requires the
display of the threshold
and baseline select by
checking the "USE"
boxes in "Show" next to
Threshold and Baseline
Amplification Plot
10 12 14 18 1fl 20 22 24 23 28 30 32 34 3S 38 «1 42 *4
Cycle
Show: 0 Threshold — D Baseline Start: Well Target^
Analysis Summary: Total Wells in ... 96 Wells
Home I [H NRSA Batch 34...032310 eds.eds x
Analysis Settings
View Well Table
Select Wells With: - Select Item -
-SelectItem- v
Show in Ta... Group By
•-I -II
-
Well
A1
A2
A3
A4
A5
A6
A7
AS
A9
10 A1D
11 A11
12 A12
13 B1
14 B2
15 B3
16 B4
17 B5
18 B6
19 B7
Omit
Flag
D
D
D
D
n
n
n
n
n
n
n
n
n
n
n
Sample... Targel
LB-123 ENT
CAL-99 25X ENT
ENT
CAL-1 00 5x SKETA
LB-123 ENT
CAL-99 25X ENT
ENT
CAL-1 00 5x SKETA
537238 ENT
CAL-1 DO 25X ENT
ENT
CAL-9825X SKETA
3. Scroll down or sideways
for more samples and
analysis parameters
a...O Scuffles
...8 Targets ...2
Disconnected
'£ start
-------
Analysis Menu- Standard Curve
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
[3 New Experiment - ,_/ Open... £J Save - Jl Close ^j Export... - t^ Print Report...
Experiment Menu«
Setup
'.*»*,
Run
" Analysis
w<- /
Amplification Plot
andard Curve
Miitticomponeirt Plot
Raw Data Plot
QC Summary
Multiple Plots View
NRSA Batch 34 testrur Standard C Rea TaaMan® Reaat I
Select "Standard Curve" to view the results of
the samples (if same target) and standards in the
plot
current settings as the default
Standard Curve
17.5 •
15.0
12.5
10.0
7.5 '
5.0
~
1 2345 10 20 30 100 200 1000 10000 10000
Quantity
Target: ENT Slope: -3.498 Y-Inter: 21.433 R*: 0.963 Eff%: 93.222
Standard •Unknown Unknown (Flagged)
Analysis Settings
w Well Table
n -
-SelectItem-
agend
1 2 3 4 5 6 7 8 9 10 11 12
2 ^ ni-.'E-"ii
c 11.. 100 S
Wells: [340 Unknown F"] 16 standard |~~] 0 Negative Control
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted Man... 0 Wells Fla.,.1 3 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
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.' start
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Analysis Menu - Standard Curve
Click here to select a different Target (if the
standards were analyzed in more than one target)
or change the Plot Color
^ Setup
Run
r Analysis
Amplification Plot
an® Reagt
Stano^rd Curve
Plot Settings \
Target
• current settings as the default
Plot Color :Default
Standard Curve
Standard curve
parameters may
be useful in data
analysis
17.5
15.0
12.5 •
10.0
7.5 •
1 2 3 45 10 2030 100 200 1000 10000 10000
Quantity
Target: ENT Slope: -3.496 Y-Inter: 21.433 R2; 0.963 Eff%: 93.222
Standard • Unknown Unknown (Flagged)
View Plate Layout | View Well Table
Select Wells With: - Select Item - v
-SelectItem-
Show in Wei...
lH View Leg end
1234567
9 10 11 12
75...
n
Wells: ^]40 Unknown Q 16 Standard [~j 0 Negative Control
Analysis Summary: Total Wells in ...96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 13 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
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Analysis Menu - Multicomponent Plot
^^^^^^^^^^^^^H
Click on the "Plot Color" drop down menu to select the
parameter based on which the graph required to be plotted
ixperiment Menu«
Setup
'.*»*,
Run
" Analysis
MI.
Amplification Plot
Standard Curve
ricomponent Plot
Raw Data Pic,
QC Summary
Multiple Plots View
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reagt
Analysis Settings
Multicomoonent Plot
efnult
Multicomponent Plot
1,100,000 -
1,000,000
900.000
800.000
111
<-> 700.000
c
2 600.000 -
in
j| 500,000
JZ 400.000
300.000
200.000
inn nnn
View Plate Layout \ View Well Table
Select Wells With:
-Select Item-
- Select Item- V
Pi Show in Wei...
TOi View Leg end
Select "Multicomponent Plot" to view the variation in the
fluorescence of the wells, targets or dyes used over the
ascending repeat of the 45 thermo cycles
5 6
8 9 10 11 12
rn57H7]p
m. 98... 100 ^
1 UU- 2
fn 63... n
1 ^S?nTia^
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39.. 4E3
-.-J ^
- 1
34.. i~n
S: 1
nknown Fj 16 Standard | ] 0 Negative Control
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted Man... 0 Wells Fla.,.1 3 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
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Analysis Menu - Raw Data Plot
Select "Raw Data Plot" to view the variation in the emission of
fluorescence of the dyes in different filters present over the
ascending repeat of the 45 thermo cycles
Analysis Settings
Setup
'.*»*,
Run
" Analysis
Amplificatir n Plot
Standard Mirve
Mutticor r>onent Plot
Raw Data Plot
QC Summary
Multiple Plots View
Raw Data Plot
Raw Data Plot
D E
Options
Show Cycle 28
! r
j I
10 15 20 25 28;
View Plate Layout 1 View Well Table
Select Wells With:
-Select Item-
- Select Item-
Show in Wei...
TOi View Leg end
1
2 ^ m .' H ."i
c 11.. 100 S
73... 100 f
en
BTl
5 6
'3 9 10 11 12
mE 98... 100
Drag the pointer on the
cycle scale to the desired
cycle number to view the
variation in the fluorescence
through filters of the
samples at that cycle
Analysis Summary: Total Wells in ... 96 Wells S... 56 Wells Omitted Man... 0 Wells Fla... 1 3 Wells Omitted by Ana... 0 Samples ...8 Targets ...2
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.' start
'* ^ B J & &
-------
Analysis Menu - QC Summary
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
New Experiment - ,_/ Open...
Save - Jl Close ^j Export... - t^ Print Report...
Experiment Menu«
Setup
'.*»*,
Run
" Analysis
Wl.
Amplification Plot
Standard Curve
Multicomponent Plot
Raw Data Plot
OC Summary
Multiple Plots \. i,»w
\
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reagt
QC Summary
Flag Summary \_
Total ... 96
Wells...56
Processe... 56 Manually Omitte... 0
Flagged... 13 Analysis Omitte... 0
Targets... 2
Sample...8
Flag Details
Fl... Name
AMP... Amplification in negative con..
BAD...
OFF...
HIG...
NO A..
NOI...
SPIKE
NOS...
Bad passive reference signal
Fluorescence is offscale
High standard deviation in re.
No amplification
Noise higherthan others in
Noise spikes
No signal in wel
•• o+ii n rni i n
F...
11
Wells
B1.B9.B1.
A1.A9
Select "QC Summary" to view the
analysis summary of the samples
in the experiment
Analysis Settings
View Plate Layout ] View Well Table
Select Wells With:
-Select Item-
-Select Item- v
Show in Wei...
j View Legend
2 ^ EI .! m.! m
-7,= 11.. 100 "•
73... 100 Z2,
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38.. 4E3
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3D.. 4E3
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_-1 ^
1
Wells: 0]40 Unknown [^ 16 Standard |~~] 0 Negative Control
. 56 Wells Omitted Man... 0 Wells Fla... 1 3 Wells Omitted by Ana... 0 Samples ... 8 Targets ... 2
Disconnected
-------
Analysis Menu - Multiple Plots View
Select "Multiple Plot View" to view all the plots for the
samples in one window
Experiment Menu«
Setup
'.*»*,
Run
" Analysis
NRSA Batch 34 testrur ...Standard C Rea... TaqMan® Reagt
Amplification Plot
Standard Curve
Miitticomponeirt Plot
Raw Data Plot
QC Summary
Multiple Plots View
Multiple Plots View
Amplification Plot - ARn... v
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Analysis - To Print a Data Report
[UiT| New Experiment -
open
Save - i_fj Close
Export...
rj) Print Report...
Experimen
Setup
Q Select data for the report. Click "Preview Report" to preview the repo
'*»*;
Run
Analysis
ontent. Click "Print Report" to send the report to the printer.
0 Results Summary
0 Plate Layout
Information about the experiment, including experiment name, experiment type, file name,user
name, run information, and comments
At;
dei
An
D Amplification Plot (ARn vs. Cycle) Da
Click on "Print Report" to make a hard
copy of the results with required plots
Standard Curv
normalized reporter (ARn) plotted against cycle number.
EH Amplification Plot (Rn vs. Cycle) Data collected during the cycling or amplification stage. Displays normalized reporter (Rn)
plotted against cycle number.
EH Amplification Plot (Civs. Well) Data collected during the cycling or amplification stage. Displays CT plotted against well
number.
Mutticomponei
Raw Data Plot
QC Summary
Multiple Plots \
0 Standard Curves
0 Results Table (By Well)
0 QC Summary
The best fit line using CT values from the standard reactions plotted against standard
quantities.
A table of experiment results for each well, including sample, target, task, quantity, ARn and CT.
Atable of flags applied to wells in the experiment, including flag description, frequency of
occurrence,and a list of flagged wells.
Print Preview |
Print Report
L
pies
Targets ...1
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* 7500 Software v2,0.1
-------
7500 Software v2.0.1
File Edit Instrument Analysis Tools Help
New Expe rime nt - ,Jj Open... A Save- Jj Close ^ Export...-
4) Print Report...
Experim*
Print Report
Setup
Run
Analysis
Q Select data for the report. Click "Preview Report' to prev
Information aboutth
•pliflcationf
Standard Curv
Multicompone
Raw Data Plot
QC Summary
Multiple Plots >
0 Experiment Summary
0 Results Summary
0 Plate Layout
0 Amplification Plot (ARn vs. Cycle)
0 Amplification Plot (Rn vs. Cycle)
0 Standard Curves
0 Results Table (By Well)
0 QC Summary
1. Select the required data and
plots to be printed by checking
the boxes next to the data and
plot types in which results can be
printed
iflllli inn ||ihmiiati
A table of experimen
dev), CT(mean) and
An illustration of the
Data collected durin
normalized reporter (ARn) plotted against cycle number.
Data collected during the cycling or amplification stage. Displays normalized reporter (Rn)
plotted against cycle number.
Data collected during the cycling or amplification stage. Displays CT plotted against well
number.
The best fit line using CT values from the standard reactions plotted against standard
quantities.
Atabie of experiment results for each well, including sample, target, task, quantity, ARn and CT.
Atable of flags applied to wells in the experiment, including flag description, frequency of
occurrence,and a list of flagged wells.
T' I llvin -
2. Click "Print Report" to
print the required data
Print Preview
T
Print Report
>i
egative Control
.8 Targets ...1
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-------
Method B
Appendix B:
Cepheid Smart Cycler® Operation
-------
-------
Method B
Appendix B - qPCR analysis on Cepheid Smart Cycler®
1.0 Smart Cycler® Operation
1.1 This protocol is intended to provide only information about critical instrument settings required to
perform EPA Method B. Further details concerning the operation of the instrument can be
obtained from the Smart Cycler® Operation Manual, Cepheid Part #D0190, Rev. D.
1.2 Turn on the Smart Cycler®; then the computer.
1.3 Double-click on the Smart Cycler® icon on the computer desktop.
1.4 The following steps for defining a protocol are only required before the initial run of the
instrument. The protocol that is defined in these steps is used in all subsequent runs of the
instrument.
1.4.1 Click on the Define Protocols icon to go to Define Protocols screen.
1.4.2 Click on the New Protocol button to open the Protocol Name? dialog. Enter "EPA
Method B" for the new protocol name, and click OK. The protocol stages are defined in
the series of boxes at the bottom of the Define Protocol screen. Make sure the new
protocol is highlighted and begin to choose state settings. To define Stage 1, click on its
drop-down box to display the menu of stage types; then select Hold. In the Temp
column, enter 50.0, and in Sees column, enter 120, leaving the Optics setting as the
default Off setting. (Note: This stage is performed to eliminate potential PCR carryover
products in the reactions using the Amp-Erase® UNG enzyme provided in the TaqMan®
Universal PCR Master Mix). Note that the optical reading cannot be programmed during
a Hold stage.
1.4.3 To define Stage 2, click on its drop-down box to display the menu of stage types, and
again select Hold. In the Temp column, enter 95.0, and in Sees column enter 600. Again,
leave the Optics setting on Off. (Note: This stage is performed to inactivate the Amp-
Erase® UNG enzyme).
1.4.4 To define Stage 3, select 2-Temperature Cycle from its drop down menu. For the first
step, enter 95.0 in the first row of the Temp column and 15 in the Sees column, Optics
column Off. For the second step, enter 60.0 in the second row of the Temp column, 120
in the Sees column, and click on the Optics cell to select On from the drop-down menu.
Note: This sets the detection of the fluorescence signal to occur at the end of the second
step in each cycle. Enter 45 in the Repeat field at the top of the Stage 3 box to specify
that it should be repeated for 45 cycles. Click the Save Protocol button.
1.4.5 To display primary curve graphs, click Define Graphs. Highlight FAM in Graph
column. Check the box for Automatically add to new runs. Under Graph Type choose
Optics from the pull down menu. Under Channels check the box for Ch 1 (FAM).
Under Show check the boxes for Primary Curve, Threshold (Vertical) and Threshold
(Horizontal). Under Axes check the box for Fluorescence vs. Cycle. At the bottom of
the screen, click on Save Graph.
1.5 Click on the Create Run icon to open the Create Run screen. For each new run, enter a unique
name in the Run Name field. (Note: The software does not allow duplicate run names).
1.6 Enter any additional information about the run in the Notes field. Click the arrow in the Dye Set
box to display a drop-down menu of the possible selections. Select FTTC25. (Note: This selects
the dye set: FAM, TET, Tex Red, Cy5, and a 25 uL reaction).
B-l
-------
Method B
1.7 Click the Add/Remove Sites button. The Select Protocols and Sites... dialog will appear.
Highlight (click on) the "EPA Method B" protocol developed prior to the first run (Section 1.4.2)
in the Protocols list. In the Sites list, highlight the sites on the instrument to be used with this
protocol in the current run by clicking on them with the shift key held down (Note: Sites refer to
the I-core modules in the Smart Cycler® processing block in which reaction tubes will be placed;
a total of 16 are possible per block. When using multiple blocks, the site numbers will be
preceded by the block letters, e.g., A, B, C); then click the right pointing arrow to transfer the
selected sites and protocol to the Selections table.
1.8 Click on the OK button to save the selections, and return to the Create Run screen.
1.9 Place the loaded Smart Cycler® reaction tubes in the I-core module slots, selected above for
current run. The tubes should snap into place. Either the front or back of the caps can face the
front of the processing block.
1.10 In one of the View menus that is shown, select Analysis Settings. The displayed table includes
one row for each of the four possible dye channels defined in the dye set. Click on the cell in the
FAM row under the Usage column heading, and select Assay from the drop down menu. Set the
Usage cells for all other dyes to Unused in the same manner. (Note: All assays in this protocol
use FAM as the reporter dye). All other cells in this table should be left at default settings (See
Smart Cycler® Operation Manual).
1.11 In the other View menu that is shown, select the Results Table. Enter the sample identification
information for each site in the Sample ID column (additional information can be entered into the
Notes column.). Leave the other columns as default settings (see Smart Cycler® Operation
Manual.).
1.12 Click on the Start Run button. The orange LEDs on the Smart Cycler® processing block should
turn on, and the software will automatically switch to the View Results screen.
1.13 To display the real time temperature profiles for all sites, click Temperature in either of the View
menus. To display real time growth curves for all samples (i.e., the fluorescence signal vs. cycle),
click FAM in the other View menu.
1.14 At the end of the run, it is recommended to check the cycle threshold values calculated by the
instrument for each sample by opening the Results Table window by clicking on this selection in
the upper View menu. It is also recommended to inspect the growth curves in the FAM window
which can be opened in the same manner from the lower View menu. The default threshold
fluorescence value is shown in this window as a single horizontal red line and the cycle thresholds
for each site are shown as vertical red lines. To view the data for individual sites in this window,
click on that site number in the table to the right of the graph. If the default threshold fluorescence
line is well above all of the growth curve lines prior to visible amplification, the threshold
fluorescence value can be changed to a lower value. This is done by reopening the Analysis
Settings window from the upper View menu and entering a new value in the Manual Thresh
Fluor Units cell in the FAM row. Conversely if the default threshold fluorescence line is below
any of the growth curve lines prior to visible amplification, the threshold fluorescence value
should be changed to a higher value in the same manner. Previous studies have indicated that a
threshold value of 8 works well for most analyses. Click on the Update Analysis button to view
the new threshold line in the FAM window. The cycle threshold values will be automatically
updated in the Results Table.
1.15 Once the threshold fluorescence value is adjusted to an optimal value, click the Save Run button.
(Note: The Smart Cycler® Software does not give a prompt to save changes before printing or
exporting. Therefore, it is possible to make changes to the Results Table or Analysis Settings, and
immediately print or export the data, then close the run without saving the changes. In this case,
B-2
-------
Method B
the data saved in the Smart Cycler® database will not match the printed or exported data. If no
changes are made in the threshold fluorescence value, the run data is automatically saved as it is
when the program is closed or a new run is created).
1.16 To set up automatic export of raw data, (see "How To Set Up Automatic Export of Raw Data" for
screen shots) from the main menu, click on Setup, then System Defaults, then Export Settings.
From the Export Settings dialog box, check the box beside Results Table and Analysis Settings.
Click the Browse button to select the folder where you want your raw data saved. Click on the
radio button next to Automatic export on run completion, then click the OK button.
1.17 To manually save the Results Table and Analysis setting containing the instrument-calculated
cycle threshold values for each sample (see "How to Manually Export Raw Data" for screen
shots), click the Export button to display the Export Data dialog box. Check the box next to the
heading Export Results Table and Analysis Settings by clicking on it. Leave all other boxes
unchecked. Click on Export. A box labeled Export Data will appear with the run name in the
file name box. Click on the drop down menu to the right of Save In: at the top left of the screen to
change the directory where you want your data saved. Always save to the hard drive. Click on
Save. Data are exported as comma-delimited text (*.cvs) files in MS Excel-compatible files to the
Export folder in the Smart Cycler® folder: C:\Smart Cycler®\Export. Analysts will late save the
file as an XLS file.
1.18 To archive a run in the Smart Cycler® program, click on Tools. From the drop down menu click
on Data Management and then Archive Runs. Click Proceed. Select the run to be archived by
clicking on its name in the database list. Click OK and then Proceed. A box labeled Archive Run
will appear on the screen. There will be a line labeled Save in:. Input which directory you would
like the run to be saved in. There will be aline File Name:. Enter a file name. Click Save. Click
OK
1.19 To print run data (see "How to Print Run Data" for screen shots), after the run is completed, right
click on the graph area, and select Print, then Print Graph with Results Table. An Optics Graph
print preview screen will pop up, click Print. The screen will switch back to the post run screen.
To print the generated report, click Report. A Run Report print preview screen will pop up, click
Print
1.20 To set up a new analysis (see "How to Set Up a New Run" for screen shots), from the post run
screen, click Create Run at the top left. The results of your just completed will disappear and an
Add/Remove Sites button will appear in the middle of the screen - click it. A Select Protocols
and Sites screen will pop up, select EPA Method B in the Protocols box, and highlight the amount
of sites needed for the assay (A1-A16) in the Sites box. Click the right arrow to the right of the
sites box to move the sights to the Selections box. After double checking that the correct protocol
is highlighted, click OK. Add the Run Name in the Run Name box. The Site ID, Protocol, Sample
ID, Sample Type, Notes (etc.) box will pop up. Enter the Sample ID for each site. Under this box,
find the Usage column and click on Assay to select Unused for Cy3, TxR and Cy5 (leave Assay
for FAM). In the same box, find the Manual Thresh Fluor Units, and click on the units for FAM
to change it from 30.0 to 8.0 (leave the units at 30.0 for Cy3, TxR and Cy5). After loading the
Smart Cycler with tubes, click Start Run. A red light should appear on sites that are in use.
B-3
-------
How to Set Up a New Experiment Using the Smart Cycler® (Software version 2.0)
Smart Cycler
User Logs Setup Tools Help
Create Run
Chech Status
Stop Run
View Results
Define Graphs
Maintenance
Protocol Name
EPA Method 1606
Ecoli EDO (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENTTaqMan Rescue
Mtd 1 607 ENT-L^C SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol-Roche TaqMan
Duplex adv to next stage example
ENTL4C-SPC EC-IC Scorpn OmniHS
demo
2 Step 60
3 Step 60 &72
rtPCR
3 Step w Melt
Fast 2 Step 60
2. This screen will appear - type in the name of the new protocol
New Protocol
Please enter a new name for the Protocol:
OK
Cancel
Stage 1
Hold ^
Temp | Sees
50.0 |120
j Optics
| Off
I
* \ .. • ,••• .-•.•.-•. :
New Protocol
Stage 2
Hold •»•
Temp | Sees | Optics
95.0 600 | Off
Delete Prc
Stage 3
Repeat j 45
\
times.
2-Temperature Cycle ^
Deg/Sec
NA
NA
Temp
95.0
60.0
Sees
15
120
Optics
Off
On
D Advance to Next Stage
i "New Protocol
| IVCIIOIUCI-IUIU^U! |
Stage 4
Unused •»
'Ceg/Sec|Temp| Sees | Optics
Stages
Unused T
Deg/Secl Tempi Sees | Optics
3. Click on OK when done
Stage
Unus
Deg/S
'••
-:
•'-•:
•':•;.
'
IM
Smart Cycler
itl - Mfcrosor.
-------
5T Smart Cycler
User Logs Setup Tools Help
Create Run
Check Status
Stop Run
View Results
Define Graphs
Maintenance
EPA Enterococcus TaqMan Method I
'
Protocol Name
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENTTaqMan Rescue
1607 ENT-LA.C SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol-Roche TaqMan
Duplex adv to next stage example
EIMTLAC-SPC EC-IC Scorpn OmniHS
demo
2 Step 60
3 Step 60 &. 72
rtPCR
3 Step w Melt
Fast 2 Step 60
1. New Protocol name will appear in list
Make sure new protocol is highlighted
and begin to choose stage settings
2. Set the Temperature in the
"HOLD" stage to 50.0 degrees and
120 seconds. NOTE optical
readings cannot be programmed
during a HOLD stage
Stage 1
Hold
Temp_ Sees | Optics
5Q.Q
1120
Off
Stage 2
Unused
Deg/SeclTempi Sees | Optics
Stage3
Unused
Deg/Secl Tempi Sees [Optics
Stage 4
Unused
Deg/Secl Tempi Sees [Optics
Stage 5
Unused
Peg/Sec] Tempi Sees [Optics
Stage
Unus
New Protocol
Delete Protocol
Duplicate Protocol
Rename Protocol
Save Protocol
luiove 10 i
op
-------
5T Smart Cycler
User Logs Setup Tools Help
Create Run
Check Status
Stop Run
View Results
Define Graphs
Maintenance
Protocol Name
EPA Enterococcus TaqMan Method
EPA Method 1606
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENTTaqMan Rescue
d 1607 ENT-LA.C SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol-Roche TaqMan
Duplex adv to next stage example
EIMTLAC-SPC EC-IC Scorpn OmniHS
demo
2 Step 60
3 Step 60 &. 72
rtPCR
3 Step w Melt
Fast 2 Step 60
In Stage 2, set the temperature to 95.0 degrees
and 600 seconds
Stage 1
Hold
Ternp
Sees
50.0
120
Optics
Off
Stage 2
Hold
Temp
95.0
Sees
600
pptics_
Off
S age 3
nused
eg/Seel Tempi Sees [Optics
Stage 4
Unused
Deg/5ec|Tempi Sees [Optics
Stage 5
Unused
DegJSeclTempi Sees [Optics
Stage
Unus
New Protocol
Delete Protocol
Duplicate Protocol
Rename Protocol
Save Protocol
Move To Top
-------
5T Smart Cycler
User Logs Setup Tools Help
Create Run
Check Status
Stop Run
View Results
Define Graphs
Maintenance
Protocol Name
EPA Enterococcus TaqMan Method
EPA Method 1606
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENTTaqMan Rescue
d 1607 ENT-LA.C SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol-Roche TaqMan
Duplex adv to next stage example
ENT LAC-SPC EC-IC Scorpn Omn
demo
2 Step 60
3 Step 60 &. 72
rtPCR
3 Step w Melt
Fast 2 Step 60
1. In Stage 3, set the drop down
menu at "2-Temperature Cycle"
to repeat 45 times (cycles).
2. For the first temp
cycle set the temp at 95
degrees for 15 seconds
with optics OFF.
3. In the second Temp
cycle, set the temp for 60
degrees for 120 seconds
with the optics ON
Stage 1
Hold
Ternp
Sees
50.0
120
Optjcs_
Off
Stage 2
Hold
Temp
Sees
95.0
600
jDp_tics_
Off
Stage 3
Repeat 45
times.
2-Temperature Cycle ^
Deg/Sec|Temp| Sees [Optics
MA 95.0 p 5 ;0ff
J12D
NA
60.0
lon
D Advance to Next Stage
Stage 4
Unused
Deg/Seel-Temp| Sees | Optics
Stage 5
Unused
DegJSeclTempi Sees [Optics
Stage
Unus
Dej/E
New Protocol
Delete Protocol
Duplicate Protocol
Rename Protocol
Save Protocol
MuuiTuTui
4. Click Save Protocol
when finished
-------
How To Set Up Automatic Export of Raw Data
Setup Tools Help
User Administration
System Defaults
Analysis Settings
Define Protocols
Define Graphs
Automatic Backup
Export Settings
NRSA 2009 Batch 25E ENT 091 809
Access Options
User: Default User
Standard - FAM
Bkgnd
Min Cycle
Bkgnd
Max Cycle
Curve Analysis
Thresh Setting
Manual Thresh
Fluor Units
Auto Thresh
#SD's
Results Table
Analysis Settings
Protocols
Primary Curve Manual
Primary Curve Manual
Standard - Tet
Primary Curve Manual
Started: Sep 18,2009 01:30 PM
Finished: Sep 18,2009 03:37 PM
Status: Done
Notes:
NRSA 2009 ENT Analysis of Batch
25E
Dye Set: FCTC25
Protocols:
Protocol
Lot Number
EPA Method 160.
Number of Sites: 30
Melt
FAM
Cy3
Texas Red
Cy5
Standard - FAM
To set up automatic export of Raw Data
into computer, click on "SETUP", then
"System Defaults" then "Export
Settings"
Save Run
Export
Report
Select Graphs
view Another Run
Delete Run(s)
Update Analysis
Import Std Curve
Compare Run
-------
Export Settings
Data Export Defaults
D Optics Data
E Results Table and Analysis Settings
D Melt Data
Optics Data Defaults
Primary Curve
2nd Derivative
1. Check Results Table and Analysis settings box n
— H
Export Path and Filename Defaults
elt Data Defaults
Melt Curve
1st Derivative
Browse
i: C:\Smart Cycler^xportWRSA 2009 CSV Files
Enter a filename to write to the same file each time.
Leave filename blank to create a unique file each time.
Filename:
2. Choose browse to
select a folder where
the raw data will export
Automatic Export Option
O No Automatic export
Automatic export on run completion
3. Be sure that Automatic export on run completion is checked
OK
4. Click OK when finished
-------
How To Manually Export Raw Data
User Logs Setup Tools Help
Create Run
Check Status
Stop Run
1
VIA
Define Protocols
nil
Define Graphs
Maintenance
Run Name:
NRSA 2009 Batch 25E ENT 091 809
User: Default User
Started: Sep 18, 2009 01:30 PM
Finished: Sep 18, 2009 03:37 PM
Status: Done
Notes:
NRSA 2009 ENT Analysis of Batch
25E
Dye Set: FCTC25
Protocols:
Number of Sites: 30
Protocol
EPA Method 160...
Lot Number
Views
Results Table
Analysis Settings
Protocols
Standard - Tet
Temperature
Intercalate
Melt
FAM
Cy3
Texas Red
Cy5
Standard - FAM
Views
Results Table
Analysis Settings
Protocols
Standard - Tet
Temperature
Intercalate
Melt
Site
ID
" '1
A2
A3
Protocol Sample ID
A4
A5
A6
A7
A8
A9
A10
A11
EPA Met.
EPA Met.
EPA Met.
. LB-96
.538480
. 538248
EPA Met..
EPA Met..
EPA Met..
EPA Met..
EPA Met..
EPA Met..
EPA Met..
EPA Met..
537352
538574
53865S
537351
538651
538655
537353
A12
A13
EPA Met..
EPA Met..
538673
538143
538045
Sample
Type
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
Notes
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
Status
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
FAM
Std/Res
FAM Ct
NEG
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
POS
0.00
35.00
35.15
34.07
39.79
36.69
37.20
33.64
28.93
29.32
33.75
36.14
36.04
Oh Dye
# Name
FAM
Assay
Cy3
TxR
Cy5
Usage
Unus...
Unus...
Bkgnd Bkgnd Bkgnd Curve Analysis
Sub Min Cycle Max Cycle
Primary Curve Manual
Thresh Setting
Manual Thresh
Fluor Units
Auto Thresh
#SD's
ON
ON
ON
Unus... ON
40
40
40
40
Primary Curve Manual
Primary Curve Manual
Primary Curve Manual
8.0
30.0
30.0
30.0
NA
NA
NA
NA
Once run is finished, click Export
Save Run
Export
| Report
Select Graphs
View Another Run
Delete Run(s)
Update Analysis
Import Std Curve
Compare Run
start
Smart Cycler
-------
8T Export Data
D Export Optics Data
Export Results Table and Analysis Settings
Export Melt Data
D Export Heater Temperature Data
^.Export
Cancel
Check the Export Results Table and Analysis Settings box
then click "Export"
-------
Export Data
Save In:
PI NRSA 2009 CSV Files ^ ^
ffl
D-D-
D-D-
D
D
>A 2009 Batch 20 STDCAL 001009
iA 2009 Batch 20A ENT 081109
iA 2009 Batch 20A SKETA 081109
iA 2009 Batch 20B ENT 081209
iA 2009 Batch 20B SKETA 001209
NRSA Z009 Batch 21 STDCAL 081709
NRSA 2009 Batch 21C ENT 082009
NRSA 2009 Batch 21C SKETA 082009
QQRSA 2009 Batch 21D ENT 082109
NRSA 2009 Batch 21D SKETA 082109
File Name:
Files of Type:
NRSA 2009 Batch 25E ENT 091 G09.csv|
All Files
Save
Cancel
Choose the appropriate file folder to save the Raw Data to
and click SAVE. NOTE: All raw data will export in the form of
a CSV file. Analysts will later save file as an "XLS" file.
-------
How to Print Run Data
§T Smart Cycler
User Logs Setup Tools Help
- n x
Create Run
Check Status
Stop Run
View Results
Define Protocols
Define Graphs
Maintenance
Run Name:
NRSA 2009 Batch 26B ENT 092409
User: Default User
Started: Sep 24, 2009 02:27 PM
Finished: Sep 24,2009 04:34 PM
Status: Done
Notes:
NRSA 2009 ENT Analysis of Batch
26B
Dye Set: FCTC25
Protocols:
Number of Sites: 32
Protocol
EPA Method 160...
Lot Number
Views
Results Table
Analysis Settings
Protocols
Standard - Tet
Temperature
Intercalate
Melt
FAM
Cy3
Texa
Cy5
Stan
Site Protocol
ID |
A1 [EPA Met...
A2 EPA Met7
A3 EPA Met...
A4 EPA Met...
A5 EPA Met...
Sample ID
Sample
Type
Notes
LB-98
537973
538019
538031
538277
UNKN
UNKN
UNKN
UNKN
UNKN
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
Status
OK
OK
OK
OK
OK
FAM
Std/Res
NEO
POS
POS
POS
POS
FAM Ct
0.00
35.31
36.99
34.12
36.54
Right click on graph area and select "Print"
then "Print Graph with Results Table"
Views
Results Table
Analysis Settings
Protocols
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Texas Red
Cy5
Standard - FAM
A12
A13
EPA Met..
EPA Met..
110
1 70+
0)
o
0)
30+
-10
-50
UNKN
UNKN
ENT5X
ENT5X
OK
OK
POS
NEO
38.46
0.00
262711
10
<1
Reset zoom
Show all sites
Scale graph
Overlay graphs
Print
Save to file (jpg)
Export graph data
Print Graph with Results Table
Site ID Protocolfsampl...
EPAMet... 537753
EPA Met...538049
EPA Met... 538050
EPA Met..537253
EPA Met..537517
EPA Met... 537143
EPA Met... 538506
EPA Met... 538496
EPA Met... 538525
EPA Met..538060
EPA Met... 538060
EPA Met... AE
EPA Met... SAE
EPA Met... 5375...
EPA Met..5375...
Save Run
Export
Report
Select Graphs
View Another Run
Delete Run(s)
Compare Run
-------
Cycler
User Logs Setup Tools Help
Create Run
Run Name:
NRSA 2009 Batch 26B ENT 09240!
User: Default User
Started: Sep 24,2009 02:27 PM
Finished: Sep 24, 2009 04:34 PM
Status: Done
Notes:
NRSA 2009 ENT Analysis of Batch
26B
Vailed amsit Cycler Customer
Optics Graph
Rim Name : NRSA 2009 Batch 26B ENT 092409
User Name : Default User
Run Date : Sep 24, 2009 02:27 PM
Protocol ISampL
Dye Set: FCTC25
Ni
This sheet
will pop up.
Click
"Print" to
print file
Al •
A3
Jl7
LB-98
537973
•^OfllC
0.00
35.31
7* OQ
EPA Method
1606 STOP
EPA Method
1606 STOP
Bali Type
Primary Cures
Lint Type
Channel
FAM
Symbol
None
B3
B5
BS
EPA Met... 537753
EPA Met... 538049
EPA Met... 538050
EPA Met... 537253
EPA Met... 537517
EPA Met...537143
EPA Met... 538506
EPA Met... 538496
EPA Met... 538525
EPA Met... 538060
EPA Met... 538060
13:' EPA Met... AE
|EPA Met... SAE
[EPA Met... 5375...
EPA Met...5375...
Save Run
Export
Report
Select Graphs
View Another Run
Delete Run(s)
Update Analysis
Import Std Curve
Compare Run
start
Smart Cycler
IB*
•-J >-v «.
-------
§^ Smart Cycler
User Logs Setup Tools Help
Create Run
Check Status
Stop Run
Define Protocols
U\
Define Graphs
Maintenance
Run Name:
NRSA 2009 Batch 26B ENT 092409
User: Default User
Started: Sep 24,2009 02:27 PM
Finished: Sep 24, 2009 04:34 PM
Status: Done
Notes:
NRSA 2009 ENT Anal
26B
Views
Results Table
Analysis Settings
Protocols
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Texas Red
Cv5
A1 [EPA Met..
A2 EPA Met..
A3 EPA Met..
Site Protocol
ID
Sample ID
LB-98
537973
'538019
A4
A5_
AS
A7
A8
A9
A10
EPA Met..
EPA Met.
EPA Met.
EPA Met.
EPA Met.
EPA Met.
EPA Met.
538031
538277
520186
520187
538579
538561
538583
Sample
Type
Notes
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
ENT5X
ENT5X.
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
ENT5X
Status
OK
OK
OK
OK
OK
OK
OK
OK
OK
OK
FAM
Std/Res
NEG
POS
POS
POS
POS
POS
POS
POS
POS
To print the generated report, click on "Report"
POS
'OS
FAMCt
0.00
35.31
36.99
34.12
36.54
31.96
34.05
31.30
27.40
Dye Set: FCTC25
Protocols:
Protocol
EPA Method 160...
Lot Number
Number of Sites: 32
Vie
Results Tfble
Analysis Settings
Protocol/
Stand art-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Red
Sta idard-FAM
OS
•JEG
32.48
26.51
38.46
0 00
Site ID| Protocol |SampL
10
20
40
Cycles
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
537753
538049
538050
537253
537517
537143
538506
538496
538525
538060
538060
WE
Export
Report
Select Graphs View Another Run Delete Run(s) Update Analysis Import Std Curve Compare Run
start
Smart Cycler
(O 11:26 AM
-------
Cycler
User Logs Setup Tools Help
Create Run
Run Name:
NRSA 2009 Batch 26B ENT 09240!
Run Report
Run Information
Rim Name: NRSA 2009 Batch 26B ENT 092409
User Name: Default User
Run Status: Done
Dye Set: FCTC25
Notes: NRSA 2009 ENT Analysis of Batch 26B
Analysis Settings:
User: Default User
Started: Sep 24,2009 02:27 PM
Finished: Sep 24, 2009 04:34 PM
Status: Done
Notes:
•JRSA2009 ENT Analysis of Batch
26B
Click "Print"
to print out
the generated
run data
report
Started: 2009^9/24 02:27 PM
Finished: 2Q09M24 04:34 PM
S W Version: 2.Od
Ch#
1
Dye
Name
FAM
Manual
Thresh
Fluor
Units
S.O
Usage
Assay
Auto
Thresh
tfSD's
NA
Bhgnd
Sub
ON
Auto Miii
Cycle
5
BkgndMin
Cycle
5
Auto Max
Cycle
10
BkgndMax
Cycle
40
Valid
Min
Cycle
3
Curve Analysis
Primary Curve
Valid Max
Cycle
60
Boxcar
Avg Cycles
0
Thresh
Setting
Manual
Taiiget
Protocols):
Name :
Lot Number:
EPA Method 1606 STOP
None
Stage 1: Hold 5Q.O°C for 120 seconds
Stage 2: Hold 95.0"C for 600 seconds
[^Temperature Cycle repeat for 45 times.
WJJCfor 15 seconds
with Optics ON
Advance to Next StajS'SWSwcles after Ch 1
Number of Sites: 32
B3
B5
B9
313
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
EPA Met...
537753
538049
538050
537253
537517
537143
538506
538496
538525
538060
538060
|AE
SAE
5375...
5375...
Save Run
Export
Report
Select Graphs
View Another Run
Delete Run(s)
Update Analysis
Import Std Curve
Compare Run
-------
How to Set Up a New Run
£T Smart Cycler
Click "Create Run"
Define Graphs
Results Table
Analysis Settings
Protocols
NRSA 2009 Batch 25E ENT 091 809
Standard - Tet
EPA Met.
EPA Met.
User: Default User
Started: Sep 18,2009 01:30 PM
Finished: Sep 18,2009 03:37 PM
Status: Done
Notes:
Standard - FAM
NRSA 2009 ENT Analysis of Batch
25E
Bkgnd Bkgnd
Min Cycle Max Cycle
Curve Analysis
Thresh Setting
Manual Thresh
Fluor Units
Auto Thresh
#SD's
Results Table
Analysis Settings
Protocols
Primary Curve Manual
Primary Curve Manual
Standard - Tet
Primary Curve Manual
Dye Set: FCTC25
Protocols:
Primary Curve Manual
EPA Method 160
Standard - FAM
Number of Sites: 30
-------
5T Smart Cycler
User Logs Setup Tools Help
Check Status
Stop Run
View Results
VIA,
Define Protocols
Define Graphs
Maintenance
Run Name:
Notes:
Dye Set:
FCTC25
Protocols:
Protocol
Lot Number
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Site ID
Protocol
Sample ID Sample Type
Notes
FAM Std
Cone
Cy3 Std TxR Std Cy5 Std
Cone Cone Cone
Click "Add/Remove" Sites
Add/Remove Sites
Q ~~
Ch Dye Usage
# | Name
FAM
Assay
Cy3
Assay
TxR
Cy5
Assay
Assay
Bkgnd
Sub
ON
ON
ON
ON
Bkgnd
Min Cycle
Bkgnd
Max Cycle
40
40
40
40
Curve Analysis
Thresh Setting
Primary Curve
Primary Curve
Manual
Manual
Primary Curve
Primary Curve Manual
Manual
Manual Thresh
Fluor Units
30.0
30.0
30.0
30.0
Auto Thresh
#SD's
NA
NA
NA
NA
Auto Min
Cycle
Auto
Cyc
10
10
10
10
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
-------
User Logs Setup Tools Help
Run Name:
Notes:
Dye Set:
FCTC25
Protocols:
Protocol
Lot Nurr
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
I~MT T-—— M —— rAf^-T C-Tf\n
EPA Enterococcus TaqMan Method
ftCPAMelliuU 1000
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENT TaqMan Rescue
Mtd 1607 ENT-LAC SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol -Roche TaqMan
A1
A2
A3
A4
AS
A6
A7
A8
Select All Sites
1. Be sure the correct
protocol is
highlighted before
selecting sites
resh
its
Auto Thresh
#SD's
Auto Min
Cycle
2. Highlight the amount of
sites needed for assay (A1 -
A16)
Cancel
OK
Auto
Cyc
I U
10
10
10
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
b EPA Draft TaqMan En.
•sWijem
-------
User Logs Setup Tools Help
U\
Run Name:
Notes:
Dye Set:
FCTC25
Protocols:
Protocol
Lot Nurr
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Select Protocols and Sites
Protocols:
Maintenance
Selections:
ENT TaqMan FAST STOP
EPA Enterococcus TaqMan Method
EPA Method 1606
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENT TaqMan Rescue
Mtd 1607 ENT-LAC SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol -Roche TaqMan
Sites:
A9
A10
A11
A12
A13
A14
A15
A16
Select All Sites
Site
Protocol
td
Cy3 Std
Cone
TxR Std
Cone
Cy5 Std
Cone
Click "right" arrow to
move sites to the
"Selections" column
Cancel
OK
Auto Min
Cycle
NA
NA
NA
NA
Auto
Cyc
10
10
10
10
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
ft TaqMan En.
Documentl - Microsof,
.^L._J.
-------
Cycler
User Logs Setup Tools Help
- n x
Run Name:
Notes:
DyeS
Protoc
Select Protocols and Sites
Protocols:
Selections:
ENT TaqMan FAST STOP
EPA Enterococcus TaqMan Method
EPA Method 1606
Ecoli BDG (uidA) TaqMan Assay
Ecoli Scorpion Assay
ENT TaqMan Rescue
Mtd 1607 ENT-LAC SPC Scorpion
Demo67
EPA Method 1606 STOP
Enterol -Roche TaqMan
Sites:
Sites have been moved to
selections column. Double
check that the correct protocol
is selected and click on "OK"
Site
A1
A2
A3
A4
A5
A7
A3
A10
A11
A12
A13
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
U\
Maintenance
Protocol
EPAEnterococcusTa.,
EPAEnterococcusTa..
EPAEnterococcusTa..
EPAEnterococcusTa.,
EPAEnterococcusTa..
EPAEnterococcusTa..
EPA Enterococcus Ta..
EPAEnterococcusTa..
EPAEnterococcusTa..
EPAEnterococcusTa..
EPAEnterococcusTa..
EPAEnterococcusTa..
EPAEnterococcusTa..
td
Cy3 Std
Cone
TxR Std
Cone
Cy5 Std
Cone
resh
its
Auto Thresh
#SD's
NA
NA
NA
NA
Auto Min
Cycle
Auto
Cyc
10
10
10
10
ft TaqMan En.
Documentl - Microsof,
•W^LIWi
-------
§^ Smart Cycler
User Logs Setup Tools Help
Check Status
Stop Run
View Results
Define Protocols
U\
Define Graphs
Maintenance
EPAEnterococcus
EPAEnterococcus
EPA Enterococcus
EPAEnterococcus
EPAEnterococcus
EPAEnterococcus
EPA Entero coccus
EPAEnterococcus
EPAEnterococcus
EPAEnterococcus
Add/Remove Sites
Bkgnd
Max Cycle
Curve Analysis
Thresh Setting
Manual Thresh
Fluor Units
Auto Thresh
#SD's
Primary Curve Manual
Primary Curve Manual
Primary Curve Manual
Dye Set:
Protocols:
Protocol
EPA Enterococcus T...
Lot Number
Graphs:
Standard-let
Temperature
Intercalate
Melt
FAM
Cy3
The Sample IDs and run name can be entered at
this point
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
start
Smart Cycler
-------
5T Smart Cycler
User Logs Setup Tools Help
Check Status
Stop Run
View Results
VIA,
Define Protocols
Define Graphs
Maintenance
Run Name:
NRSA 2009 Batch 1 SKETA 092209
Notes:
NRSA 2009 SKETA Analysis of Batch 1
Dye Set:
FCTC25
Protocols:
Protocol
EPAEntercoccus qP...
Lot Number
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Cy3
Site ID
Protocol
A1
A2
A3
A4
A5
A6
A7
AS
A9
A10
EPAEntercoccus q..
EPAEntercoccus q.
EPAEntercoccus q..
EPAEntercoccus q..
EPA Entercoccus q.
EPA Entercoccus q.
EPAEntercoccus q..
EPAEntercoccus q..
EPAEntercoccus q..
EPAEntercoccus q..
Entercoccus
Sample ID Sample Type
LB-1
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
Notes
SKETA 5X
SKETA 5X
SKETA 5X
SKETA 5X
SKETA 5X
SKETA 5X
SKETA 5X
SKETA5X
SKETA5X
SKETA 5X
SKFTA fiXl
Add.'Remove Sites
Ch
#
Dye
Name
il [FAM
Assay
Cy3
TxR
Cy5
Usage
Assay
Ass^y
Assay
Bkgnd
Sub
ON
ON
ON
ON
Bkgnd
Min Cvcle
Bkgnd
Max Cvcle
40
40
40
40
Curve Analysis
Thresh Setting
F^imary Curve.
Primary Curve
Manual.
Manual
Primary Curve
Primary Curve Manual
Manual
Manual Thresh
Fluor Units
Auto Thresh
#SD's
Auto Min
Cvcle
Auto
Cvc
30.0
30.0
30.0
30.0
NA
NA
NA
NA
10
10
10
10
Under the "Usage" column, select "Unused" from
the drop down menu for Cy3, TxR and Cy5. Leave
the "Assay" on the FAM line for this particular
protocol
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
-------
§^ Smart Cycler
User Logs Setup Tools Help
Check Status
Stop Run
View Results
Define Protocols
U\
Define Graphs
Maintenance
Run Name:
NRSA2009 Batch 1 SKETA 092209
Notes:
NRSA2009 SKETA analysis of Batch 1
Dye Set:
FCTC25
Protocols:
Protocol
EPAEnterococcusT...
Lot Number
Graphs:
Standard-Tet
Temperature
Intercalate
Melt
FAM
Icy3
Site ID
A1
A2
A3
A4
A5
A6
A7
AS
A9
A10
A11
Protocol
EPAEnterococcus...
EPA Enterococcus .
EPAEnterococcus.
EPAEnterococcus.
EPAEnterococcus.
EPAEnterococcus.
-::-••-•••
AddjRei
Sample ID
LB-1
Sample Type
UNKN
UNKN
UNKN
UNKN
UNKN
UNKN
Notes
SKETA 5X
SKETA 5X
SKETA5X
SKETA5X
SKETA 5X
SKETA5X
1. Set the Manual Threshold
Fluorescence Units to 8.0 on the
FAM line
Ch
#
Dye
Name
FAM
Assay
Cy3
TxR
Cy5
Usage
Unus...
Unus...
Unus...
Bkgnd
Sub
ON
ON
ON
ON
Bkgnd
Min Cycle
Bkgnd
Max Cycle
40
40
40
40
Curve Analysis
Thresh Settinc
Primary Curve Manual
Primary Curve Manual
Primary Curve Manual
Primary Curve Manual
Manual Thresh
Fluor Units
8.0
30.0
30.0
Auto Thresh
#SD's
NA
NA
NA
NA
Auto Min
Cycle
Auto
Cyc
10
10
10
10
2. When smart cycler tubes have been put in to
appropriate sites, click "Start Run" - a red light
should appear on sites that are in use
Start Run
Cancel Run Setup
Report Run Setup
Select Graphs
Copy Run Setup
start
Smart Cycler
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