EPA/600/A-93/013
ISOLATION AND PURIFICATION OF BACTERIAL DNA
FROM SOIL
William E. Holben
Center for Microbial Ecology and Department of Crop and Soil Sciences
Michigan State University
This is a preprint of a chapter to be published in "Methods of Soil Analysis"
published by the Agronomy Society of America, Inc.; 1992 (in press)
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ISOLATION AND PURIFICATION OF
BACTERIAL DNA FROM SOIL
William E. Holben
Center for Microbial Ecology and Department of Crop and Soil Sciences
Michigan State University
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INTRODUCTION
Recently, new methods for monitoring specific bacterial populations8 in
environmental samples have become available. These methods employ the
techniques of molecular biology to distinguish, enumerate and monitor individual
bacterial populations within a microbial community by the detection of DNA
sequences specific to those populations using appropriate molecular probes.
DNA-based detection of bacterial populations helps to overcome a major limitation
of microbial ecology and soil microbiology; the difficult task of specifically
monitoring an individual population of microbes in the environment, and in the
presence of the entire microbial community. Such capabilities are essential to
understanding the complex interactions between the environment, other
microorganisms and the population(s) of interest. Prior to the development of
these methods, microbial ecologists generally monitored specific microbial
populations using methods that included developing mutant derivatives that
could be recovered on selective media (e.g., spontaneous antibiotic resistance), or
polyclonal or monoclonal antibodies raised against individual populations that
were conjugated to a Quorecent dye to facilitate detection by direct microscopic
analysis. DNA-based detection of microbial populations thus represents a new
tool to expand the capabilities of investigators to detect and quantify
microorganisms in environmental samples.
Among the advantages of DNA-based microbial monitoring methods are
that a particular DNA sequence is detected directly; thus gene expression is not
required. Marker genes and selectable phenotypes are also not required. This is
potentially important to microbial ecologists in that it obviates the need to
a For the purposes of this chapter, an individual population of bacteria is defined as a group of
organisms having identical, or nearly identical, genotype (genetic makeup).
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demonstrate that genetic manipulations of organisms required for detection by
alternate strategies have not compromised the competitiveness of the organism.
Thus, DNA-based detection methodologies can be used for monitoring either
genetically engineered or wild-type indigenous populations. In fact, very little
information about the genetic make-up of the population of interest is required
since highly specific probes for detection can be generated by simply subcloning
random pieces of DNA from the organism of interest and screening for specificity
(Salyers et al., 1983). Alternatively, the probe sequence can be based on regions of
the rRNA gene(s) specific to the population of interest which can readily be
identified (Barry et al., 1990). Another advantage of DNA-based detection is that
bacterial growth is not required for detection allowing non-culturable and non-
viable populations to be detected. The importance of this aspect is clear when one
considers that typically only about 0.1-1 percent of the bacteria present in a soil
sample can readily be cultured under laboratory conditions (Faegri et al., 1977).
Having no requirement for culturing also simplifies quantitative and comparative
analyses since there is no subsequent increase in the population(s) of interest
compared to the other microbial populations in the community and thus relative
proportions between populations are maintained.
As with any other method for microbial detection, there are limitations to
these molecular approaches. Limitations do not preclude the use of this or any
other detection strategy, they simply need to be recognized and understood. This
will help to avoid ambiguities and potential over-interpretation of data or
overestimation of detection capabilities. For example, when using probes for
specific functions (e.g., a certain catabolic activity), lack of hybridization signal
can only be construed as absence of the sequence used as probe, not absence of the
activity. One potentially limiting aspect of DNA-based detection strategies is that
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protocols for DNA isolation, purification, and subsequent detection and
enumeration are relatively sophisticated compared to simpler detection strategies
such as culturing on selective media. Relatively few samples (usually 6-24) can be
simultaneously processed. This might limit some ecological studies particularly
where a large number of variables are to be studied or where statistical analyses
are required. The low-end sensitivity of DNA-based detection strategies is
relatively poor. Without incorporating additional sophisticated protocols that
amplify either the target sequence (e.g., Steffan and Atlas, 1988; Neilson et al.f
1992) or the probe signal, DNA probes can detect about 104 copies of target
sequence per gram of soil (Holben et al., 1988). Since there are generally about 109
bacteria in a gram of surface soil, this represents the detection of populations that
constitute about 1/100,000 of the total microbial community. This level of detection
might be a limitation for some analyses, e.g., risk-assessment for engineered
organisms, but certainly suffices for many other kinds of investigations where
one is interested in more numerically dominant populations. Recent
improvements in DNA isolation protocols and subsequent analyses have made
these procedures simpler and more generally useful than as originally published.
Two alternate strategies for the isolation of total bacterial community DNA
from soil samples will be presented; the first is based on the fractionation of
bacteria from soil prior to lysis and the second involves direct lysis of bacteria in
the presence of the soil matrix, A comparison of these methodologies is made,
and recommendations for the selection of either protocol, depending on sample
characteristics and the experimental question being addressed, are given. Other
chapters of this volume describe in detail some of the analyses and potential
applications possible with bacterial community DNA. For an overview of this
emerging DNA-based microbial detection technology, the reader is referred to
Holben and Tiedje (1988); Sayler and Layton (1990); and Knight et al. (1992).
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GENERAL CONSIDERATIONS
There are several considerations that will affect the recovery of total
bacterial community DNA from the soil environment, regardless of which of the
two DNA recovery methods are employed.
Biomass
The bacterial biomass present in the sample has an impact on the quantity
of DNA recovered due to some practical limitations of the methodologies
employed. Theoretically, one could recover bacterial DNA from each bacterium
present if every important reaction and handling step operated at 100% efficiency.
Then, the starting sample size could be adjusted to obtain the desired yield of
DNA. This, of course, is not the case. For example, with the direct lysis protocol
described here, the starting sample size has been scaled down to 10 g (compared to
the original protocol of Qgram et al. [1987], where 100 g samples were used) in
order to increase the number of samples that can be processed simultaneously.
Using the direct lysis protocol outlined below, 6-10 ng of bacterial community DNA
per gram of soil can routinely be obtained for a surface soil (A horizon) that
contains about 109 bacteria per gram, for a yield of about 60-100 ug per sample.
However, the number of bacteria per gram of soil varies with soil type and depth.
At a depth of 1 meter, where microbial populations typically range from 106 to 10?
bacteria per gram, one would need to process on the order of 1 kg of soil to obtain a
yield of 100 ug of bacterial community DNA. Samples of this size are not readily
be processed by the method outlined below. The procedures, as given, are
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intended for use with samples such as surface soils, sediments, or sludges with
total bacterial counts in the range of 108 to 101° bacteria per g of material. Others
have developed modifications of the direct lysis protocol in order to recover useable
amounts of DNA from samples with low biomass such as aquifer material (S.
Thiem, personal communication; Smith and Tiedje, 1992).
Organic/Humic content of soils
The amount of organie/humic matter in soil has a dramatic effect on the
quality (purity) of the DNA obtained, particularly when the direct lysis method is
employed. The humic materials present in soil have a similar molecular weight
and net charge to DNA and, thus, are readily copurified. The bacterial
fractionation procedure which first separates bacteria from the bulk soil prior to
cell lysis reduces, but does not eliminate, this problem. Humic contaminants
interfere with subsequent enzymatic digestions of DNA (Ogram et al. 1987; Holben
et al., 1988; Steflan et al,, 1988), and potentially other enzymatic reactions such as
DNA polymerase and ligase. Humic contaminants also confound precise
quantitation of the recovered DNA because they exhibit substantial absorbance of
light at 260 nm, the measure of which is generally used to quantitate DNA. It is
thus preferable to use soils of moderate organic/humic content for experiments
involving the isolation of total bacterial community DNA. Although there may be
a correspondingly lower biomass associated with such soils, it is usually
sufficient to recover usable amounts of DNA from surface soils. For cases in
which high organic content soils are used, methods for more precise quantitation
of DNA in the presence of humic contaminants are available. One such method,
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which is relatively simple and requires no sophisticated equipment, is outlined
later in this chapter.
Clay Content of Soils
Adsorption isotherms indicate that relatively large amounts of DNA can
bind to pure clays, soils and sediments (Greaves and Wilson, 1969; Ogram et al.,
1988). The binding of DNA to clays in soil can have a profound impact on the
amount of bacterial community DNA recovered. For example, in comparing
DNA recovered from two soils with similar organic content (2.3 and 2.7%
respectively), but different clay content (8.1 and 48% respectively), it was found
that the yield of DNA from the high clay soil was only about 15-25% of that from
the low clay soil despite both soils having similar bacterial viable counts
(unpublished observation). The mechanism of binding of DNA to clay is not well
understood. Studies with flavomononucleotide binding to smectite implicate the
Fe3+ groups of clay and the phosphate groups of the mononucleotide (Mortland, et
al., 1984). However, attempts to block DNA binding to clays in soil by competition
with excess phosphate, or altering the pH or ionic environment, have been largely
unsuccessful (unpublished data). Thus, no method for effectively blocking the
DNA binding sites of clays in soil, or for removing adsorbed DNA from clays (and
thus enhancing DNA recovery) has yet been described. As was the case with high
organic matter soils discussed above, it is best, if possible, to avoid using high clay
soils in experiments where bacterial community DNA is to be recovered.
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BACTERIAL FRACTIONATION METHOD FOR RECOVERY OF
BACTERIAL COMMUNITY DNA
Principles
The bacterial fractionation method involves the separation of bacterial cells
from the bulk of the soil prior to cell lysis and recovery of bacterial community
DNA. Briefly, soil particles, debris, fungal cells and bacterial cells are brought
into suspension by homogenization in the presence of buffer. Soil particles,
fungal cells, and other debris are then removed by a low-speed centrifugation step
which leaves the unattached bacterial cells in suspension. High-speed
centrifugation is then performed to recover the bacterial cells. This combined
low-speed, high-speed centrifugation method for the recovery of bacterial cells
from soils was pioneered by Faegri et al. (1977) and is termed differential
centrifugation. Generally, multiple rounds of homogenization and differential
centrifugation are performed on the same soil or sediment sample to enhance the
recovery of bacteria. In the protocol described here, the bacterial fraction is lysed
using a protocol which combines the salient features of lysis protocols for various
groups of bacteria, including the removal of humic contaminants using
polyvinylpolypyrrolidone (PVPP), digestion by lysozyme and pronase, and
incubation at high temperature (Holben et al., 1988). Following cell lysis, the
bacterial DNA is recovered and purified by cesium chloride-ethidium bromide
equilibrium density centrifugation and precipitation in ethanol. As described
here, the method includes several modifications of the previously published
protocol (Holben et al., 1988) including the deletion and combination of steps to
result in a shortened protocol that gives comparable yields and purity of DNA.
The yield of DNA from 50 g of agricultural surface soil with 2.3% organic matter
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and 8.1% clay content is in the range of 50-100 ug, corresponding to 1-2 ug per
gram of soil. This protocol allows bacterial community DNA to be purifed from
six 50 g soil samples to be processed to the point of purified bacterial community
DNA in 3-4 days. With slight additional effort, it is possible to process up to 12
samples simultaneously. The bacterial fractionation and cell lysis portion of the
protocol (i.e., "day 1" of the procedure) is labor-intensive but the subsequent
purification of DNA is largely "hands-ofT time.
Advantages
An advantage of the bacterial fractionation method is that the DNA
recovered from soils, especially high organic content soils, tends to be less
contaminated with humic materials than is DNA recovered by direct lysis since
the bacteria are removed from the bulk of the soil prior to lysis. If the analysis of
the bacterial community DNA isolated from a particular soil requires subsequent
digestion with restriction endonucleases and the organic content of the soil is
high, isolation of DNA by bacterial fractionation may be required. The DNA
recovered by this method has an average size of 50 kb (Holben et al., 1988)
compared to DNA recovered by direct lysis which ranges from 30-40 kb in size
(unpublished observation). This might be a consideration if the recovered DNA
were to be used in experiments attempting to clone DNA fragments from the
bacterial community DNA but is not as important when the DNA is to be used in
hybridization experiments. The bacterial fractionation procedure recovers DNA
only from bacteria (not fungal, protozoan or free DNA) since the bacteria are
separated from the soil prior to lysis and DNA recovery. Initially, this appeared to
be an important feature of this protocol (Holben et al., 1988; Steffan et al., 1988), but
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more recently it appears that the direct lysis protocol also recovers primarily
bacterial DNA (see below). Originally (Holben et al., 1988), it was thought that the
DNA recovered by the bacterial fractionation method was representative of the
entire bacterial community based on indirect evidence (Bakken, 1985), but more
recent data from this laboratory indicate that more recently grown cells are
preferentially recovered from the soil environment (manuscript in preparation).
Bacterial fractionation may preferentially recover rapidly growing bacterial cells
because they do not adhere as tightly to soil particles. This can be used to
advantage if the bacterial population of interest is rapidly growing; providing a
fractionation of actively growing cells from those less active, thereby increasing
the sensitivity of detection of the desired population. Another advantage of the
bacterial fractionation method is that it yields viable cells from the soil bacterial
community in concentrated form (Holben et al., 1988) which can be used in other
types of experiments requiring live bacteria. The bacterial fractionation protocol,
as described here, recovers about 33% of the bacterial cells from sandy loam soil
after three rounds of homogenization and differential centrifugation (Holben et
al., 1988). Additional rounds of homogenization and centrifugation will yield a
diminishing return of bacterial cells for the effort involved, but may be necessary
to recover more tightly adhered bacterial cells.
Disadvantages
Perhaps the main disadvantage of this procedure is that it requires a
significant amount of "hands-on" activity and time to process samples through
the point of cell lysis (typically 6-8 h for six samples). Although there are no
particularly sophisticated procedures or specialized equipment involved, care
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must be taken in handling samples to minimize DNA loss and maximize
reproducibiiity of yield. Due to the amount of handling involved, and the
limitations of centrifuge rotor configuration and other logistical considerations, it
is usually practical for a single person to process six soil samples simultaneously
for bacterial community DNA,
although up to 12 samples per day can be accommodated. The bacterial
fractionation protocol yields 5-6 times less DNA per gram of soil than does the
direct lysis procedure. This can be an important consideration, particularly
when the samples have low bacterial biomass. As mentioned above, this
procedure appears to preferentially recover DNA from rapidly growing and less
tightly adhered bacteria. This phenomenon may confound attempts to quantify
bacterial populations unless hybridization data are compared to independant
measurements using alternate methodologies (Holben et al., submitted). This
selectivity would be a disadvantage for experiments in which the objective is to
obtain DNA representing the entire bacterial community, for example to assess
the diversity of organisms present in the community that contain a certain gene
or other target sequence.
DIRECT LYSIS METHOD FOR THE RECOVERY OF TOTAL
BACTERIAL COMMUNITY DNA.
Principles
In the direct lysis method, bacterial cells are lysed directly in the presence
of the soil matrix. High temperature, high concentrations of detergent and
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mechanical disruption using minute glass beads are employed for cell lysis using
this method. Direct lysis of bacterial cells in soil for the recovery of total bacterial
DNA was pioneered by Ogram et al. (1987) and has since been modified by several
groups for simplification, increased sample throughput, or specific applications
(Steffan et al., 1988; Hilger et al., 1991; Tsai and Olson, 1992; Smith et al, 1992;
this manuscript). As originally described, one or two 100 g soil samples could be
processed for DNA recovery over the course of 3-4 days and several DNA
concentration/precipitation steps were involved. In the protocol described here,
the released DNA is subsequently isolated and purified by cesium chloride-
ethidium bromide equilibrium density centrifugation and precipitation in
ethanol. The time required to obtain purified DNA is 2-3 days which represents
an improvement over the original protocol, is less labor-intensive, and greater
numbers of samples (12-24) can be processed simultaneously.
Advantages
A major advantage of the direct lysis approach is that it is less labor
intensive and faster than the bacterial fractionation method. The direct lysis
protocol allows one person to readily process 8-12 samples simultaneously, and up
to 24 samples with extra effort. The time required from the starting point of sieved
soil samples to the initiation of cesium chloride gradient centrifugation is only 2-3
h. The direct lysis procedure results in higher yields of DNA, typically 60-100 jig
per 10 g soil sample (6-10 ng pef g of soil). This is particularly important in
samples having low biomass such as aquifer material where the ability to recover
sufficient amounts of DNA can be the limiting factor to success. Bacterial
community DNA recovered by direct lysis seems to better represent the bacterial
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community than DNA recovered by the bacterial fractionation procedure
(manuscript in preparation). In fact, it appears that the direct lysis protocol
outlined here approaches quantitative recovery of bacterial DNA since there are
about 109 bacteria per gram of soil, each having a genome of about 9 x 10-15 g of
DNA, resulting in a calculated quantitative yield of 9 \ig DNA per gram of soil. It
is also significant that essentially no intact bacterial cells can be found by
microscopy in a soil sample following the lysis protocol. This is an important
feature for experiments involving populations that are tightly adhered to
particles, not rapidly growing, or when DNA representative of the entire bacterial
community is of interest.
Disadvantages
Bacterial community DNA isolated by the direct lysis procedure is generally
more contaminated with humic material than is DNA isolated from the same soil
by the bacterial fractionation method. However, DNA purified by direct lysis from
soils having moderate levels of humic acids is generally of sufficient purity for
most subsequent manipulations involving digestion with restriction enzymes,
denaturation, or hybridization. If particularly fastidious reactions, such as DNA
amplification using the polymerase chain reaction (PCR), are to be performed, or
if DNA isolated using this method is refractory to restriction digestion or other
manipulations, further purification (e.g., by additional rounds of purification on
cesium chloride-ethidium bromide gradients), or isolation of DNA by bacterial
fractionation may be required. DNA isolated by direct lysis tends to be oflower
molecular weight (i.e. the randomly sheared DNA fragments are, on the average,
smaller) than DNA isolated by bacterial fractionation. Presumably, this reflects
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the more harsh conditions (i.e. higher temperatures and ionic detergents) and
mechanical shearing of DNA imposed by this procedure. The average DNA
fragment size is still over 25 kb and thus is suitable for most procedures involving
size-fractionation of restriction enzyme-digested DNA, It is worthwhile to assess
the size range of DNA obtained by this method since excessive shearing will yield
smaller DNA and less satisfactory results in hybridization analyses, or if
attempting to clone DNA from the microbial community. Size range can be
readily determined by agarose gel electrophoresis with DNA fragments of known
size included as standards.
As mentioned above and elsewhere (Ogram et al., 1987; Steffan et al., 1988),
it was thought that, in addition to bacterial DNA, the direct lysis procedure might
recover DNA from fungi, protozoa, and cell-free DNA. Recent evidence suggests
that these other potential sources of DNA in soil do not contribute significantly to
the DNA obtained. Other investigators have made concerted attempts to obtain
fungal DNA from soil samples with little success using the direct lysis method
and similar approaches (D. Harris, personal communication). It seems likely
that the rigid fungal cell wall and the prevalence of "ghost" fungal hyphae
containing no DNA account for the difficulty in isolating DNA from fungi. The
population levels of protozoa in typical agricultural surface soils are about 105 per
gram compared to 109 to 10*0 bacteria. Thus, even accounting for the larger
genome size of protozoa, they could not make a significant contribution to the total
amount of DNA obtained. Free (extracellular) DNA in the agricultural soils used
in this laboratory, if present, does not appear to be extracted by the direct lysis
protocol. This is evidenced by the fact that initial extraction of the soil for DNA
prior to lysis does not recover DNA; nor does it reduce the DNA yield obtained by
direct lysis compared to soil not previously extracted. In sediment samples, the
initial extraction of DNA prior to cell lysis recovered about 1 ug of DNA per g of
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sediment compared to the recovery of 26 |ig of DNA per g of sediment following
cell lysis (Ogram et al., 1987).
In summary, the direct lysis procedure is simpler, faster and gives higher
yields of DNA that is probably more representative of the total bacterial
community present in the soil sample than DNA obtained by the bacterial
fractionation method. On the other hand, DNA obtained by bacterial fractionation
is of higher purity, larger molecular weight and enriched for rapidly growing
(and thus, the most active) populations. The method chosen for use thus depends
on the nature of the experimental question, the requirements of the subsequent
analyses to be performed with the bacterial community DNA, and the
characteristics of the environmental sample. Some recommendations for the
appropriate bacterial community DNA recovery method based on experimental
goals or soil characteristics are given in Table 1.
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Table 1. Considerations and recommendations for selection of the appropriate
protocol for isolating bacterial community DNA from soil.
Consideration
low biomass
high purity of DNA
large number (#) of
samples
high organic/humic
content of samples
rapidly growing or
loosely adhered orgsc
tightly bound orgs
DNA best represents
community
Example
aquifer material or
nutrient poor soil
for PCR reactions
multi-parameter or
high replication
rich soils, forest
litter
addition of specific
source of carbon
EPSd producers or
trait of organism
assess diversity or
community-level
analyses
Protocol
DLa
BFb
DL
BF
BF
DL
DL
Reason(s)
better DNA recovery
and low humic
matter
bacteria removed
from soil before lysis
faster, larger
sample #'s possible
bacteria removed
from soil before lysis
enriches for these
orgs; > sensitivity
lyses adhered cells
probably more
representative
aDL = Direct Lysis protocol
t>BF= Bacterial Fractionation protocol
corgs = organisms
dEPS = exopolysaccharide
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PROCEDURES
L Bacterial Fraetionation
Materials
NOTE: The materials list is based on the simultaneous processing of six 50 g soil
samples from the stage of sieved soil to the final cesium chloride gradient
purification step. The list is based on the items used in this laboratory and does
not constitute a commercial endorsement of any supplies or equipment.
Reasonable substitutions for particular types of centrifuge rotors, tubes and other
materials and equipment that maintain, or reasonably approximate, the specified
conditions are appropriate.
1. Six standard Waring blenders with 1.2 1 (40 fi. oz.) glass jars
2. Ice/water bath for each of the blender jars
3 Twelve 250 ml centrifuge bottles (for use with a Sorvall GSA rotor)
4, Sorvall superspeed centrifuge (e.g., model RC5B) with an SS34 (8-place) or
SAGOO (12-place) rotor and a GSA rotor
5. Six small paint brushes (to facilitate resuspending bacterial pellets)
6. Twelve 50 ml polycarbonate Oak Ridge tubes
7. Vortex mixer
8. Water bath at 37° C
9. Water bath at 65° C
10, Refractometer (to measure the initial density of the cesium chloride gradient
for DNA purification. If a refractometer is not available, density can be
determined by measuring the specific gravity of the solution).
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11. Sorvall ultracentrifuge with TV865B rotor or equivalent
12. Twelve ultracentrifuge tubes for the TV865B rotor
Recipes
NOTE: The recipes presented here are for reagents required from the stage of
sieved soil to the point of the final equilibrium gradient centrifugation step. After
this point the steps are common to both DNA isolation protocols and can be found
in the section titled: "Fractionation of DNA Gradients, Final Purification and
Quantitation of Bacterial Community DNA".
1. 10 X Winogradsky's Salt Solution ( 10 X WS)
Provides an isotonic environment for bacterial cells during homogenization
and differential centrifugation steps.
for 2 liters:
•Dissolve 5.0 g of K2HP04 in 800 ml of distilled H20.
•Dissolve the following in (a separate) 800 ml of distilled H20:
MgS04 • 7 H20 5.0 g
NaCl 2.5 g
Fe2(S04)3 • H20 50 mg
MnSC>4 * 4 H2° 50
•Combine the above, then adjust to pH 6.0 with concentrated HCl.
•Bring the final volume to 2 1 with distilled H20.
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•Before use, dilute 1:10 with distilled H20 and then autoclave.
2. Homogenization solution*
This solution provides an isotonic environment for bacterial cells and
contains ascorbic acid as a reducing agent to prevent further oxidation
(which results in polymerization) of humic compounds in the soil.
Contains:
1 X Winogradsky's salt solution
0.2 M sodium ascorbate (added as powder to achieve 0.2 M)
This should be prepared just prior to use as the reducing power of the
sodium ascorbate lessens with time.
NOTE: If the bacterial fractionation protocol is being used to recover viable
cells, it is recommended that Winogradsky's salt solution alone be used as
the homogenization solution as sodium ascorbate appears to reduce the
viability of cells during the fractionation process.
3. Acid-washed Polyvinylpolypyrrolidone (PVPP)
This insoluble polymer complexes with humic acids in the homogenization
stage removing them from the aqueous phase. The acid treatment of PVPP
constitutes a pretreatment which optimizes this interaction.
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•Prepare 4 1 of 3 M HC1. (Most stock concentrated HCl solutions are 12.1
N so slowly add 992.0 ml of concentrated HCl to 3008 ml of distilled H20
in a 4 1 beaker).
•Slowly add 300 g of polyvinylpolypyrrolidone with stirring, cover beaker
and stir overnight.
•Filter suspension through Miracloth or several layers of cheesecloth
(use a large Buchner funnel and a 4 1 vacuum flask).
•Resuspend the PVPP in 4 1 of distilled H20, mix for 1 hour and again
filter through Miracloth or cheesecloth.
•Resuspend the PVPP in 4 1 of 20 mm potassium phosphate buffer (pH
7.4) and mix for 1 to 2 h. Check the pH of the PVPP suspension with pH
paper. The desired pH is 7.0.
•Repeat filtrations of the suspension and washes in 20 mM phosphate
buffer until the PVPP suspension has a pH of 7.0.
•Following the final filtration, spread the PVPP on lab paper and let air
dry overnight.
4. TE
Protects DNA by providing buffered environment with EDTA present to
chelate divalent cations which are required for the activity of any nucleases
which might be present.
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Contains:
33 mM Tris, pH 8.0
1 mM EDTA, pH 8.0
for 2 1:
•Combine the following in 1 1 of distilled H20:
66 ml of 1 M Tris, pH 8.0
4 ml 0.5 M EDTA, pH 8.0
•bring volume to 2 1, then autoclave.
NOTE: The disodium form of EDTA is preferred. At high concentrations
EDTA will not go into solution until it approaches the appropriate pH.
5. 5 M sodium chloride (NaCl)
Sodium chloride is used during the cell lysis stage as a pretreatment for
cells with exopolysaccharide capsules to facilitate access of lysozyme to the
cell wall for more efficient lysis.
for 500 ml:
•Add 146.1 g of NaCl to 400 ml distilled H20 and dissolve with stirring.
•Bring the volume to 500 ml, then autoclave.
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6. 20% Sarkosyl
This detergent disrupts the membranes of bacterial cells facilitating the
release of DNA into solution during the cell lysis stage.
for 100 ml:
•Add 20 g of n-laurylsarcosine to 50 ml of distilled H20, mix (slight
heating will help the sarkosyl go into solution)
•Bring the volume to 100 ml, then autoclave.
7. Tris/sucrose/EDTA
This solution provides an appropriate environment for bacterial cells that is
buffered both for pH and for osmotic potential and inactivates endogenous
nucleases (which degrade DNA) by chelating divalent cations which are
required for their activity. The EDTA also serves to disrupt the outer
membrane of Gram negative organisms allowing lysozyme freer access to
the cell wall.
Contains:
50 rnM Tris, pH 8.0
0.75 M Sucrose
10 mM EDTA, pH 8.0
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for 250 ml:
•Combine the following in 200 ml of distilled H20:
12.5 ml of 1 M Tris, pH 8.0
64.2 g sucrose
5.0 ml of 0.5 M EDTA, pH 8.0
•Bring volume to 250 ml with distilled H20, then autoclave.
8. Lysozyme solution (40 mg/ml)
Lysozyme enzymatically attacks the cell wall of bacteria allowing rupture of
the cell membrane by detergent and the release of DNA into solution.
for 5 ml:
•Dissolve 200 nig of lysozyme (grade 1 from chicken egg white [Sigma
#L6876]) in 5,0 ml of TE. Prepare on same day and store on ice until use.
9. Pronase E (10 mg/ml)
Pronase E comprises a mixture of protein degrading enzymes that facilitate
the rupture of bacterial cells in the lysis stage.
for 5 ml:
•Dissolve 50 mg of pronase (type XXV from Streptomyces griseus [Sigma
#P6911]) in 5 ml of TE. Preincubate for 30 min at 37° C prior to use to
allow the proteases to inactivate any contaminating nucleases in the
mixture.
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10. Ethidium bromide (10 mg/ml)
This DNA-binding dye intercalates into DNA molecules and imposes
changes in the buoyant density of DNA causing it to form a discrete band in
the cesium chloride equilibrium density gradients that can be fractionated
and further purified.
for 100 ml:
Dissolve 1 g of ethidium bromide in 100 ml of TE. Overnight mixing with
a magnetic stirrer may be required.
NOTE: ethidium bromide is a potent mutagen and should be handled
with care.
11. Cesium chloride balance solution (Rf = 1.3885)
This solution is used to bring cesium chloride gradient tubes to final
volume and to balance the tubes prior to ultracentrifugation.
for approximately 300 ml:
•Add 250 g of finely ground cesium chloride (CsCi) to 250 ml of distilled
H20 (sterile) and mix by inversion until the CsCl is dissolved.
•Add 12.5 ml of 10 mg/ml ethidium bromide and mix.
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'Check the refractive index (Rf) and adjust as necessary to achieve an Rf
value of 1.3885 (this corresponds to a density of 1.58) by adding CsCl to
increase the refractive index (density) or H20 to decrease the refractive
indexi
NOTE: As mentioned above, refractive index is a measure of the density
of the solution. In lieu of using a refractometer the investigator can
determine the specific gravity of the solution and adjust the density of
the solution to 1.58.
Procedure
NOTE: The following protocol describes the steps involved from the stage of sieved
soil to the point of the final equilibrium gradient centrifugation step. After this
point, the steps are common to both DNA isolation protocols and can be found in
the section titled: "Fractionation of DNA Gradients, Final Purification and
Quantitation of Bacterial Community DNA".
1. Combine each 50 g soil sample with 200 ml of homogenization solution and 15
g of acid-washed polyvinylpolypyrrolidone (PVPP) in a blender jar.
2. Homogenize for three 1 min intervals with 1 min cooling in an ice/water bath
between homogenizations.
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26
3. Pour the homogenate into 250 ml centrifuge bottle and pellet soil, fungi and
other debris by centrifugation in a Sorvall GSA rotor at 2,500 rpm (640 x g) for
15minat4°C.
4. Carefully pour the supernatant into a clean 250 ml centrifuge bottle and
collect the bacterial fraction by centrifugation in a Sorvall GSA rotor at 12,000
rpm (14,740 x g) for 20 min at 4° C.
5. Add 200 ml of homogenization buffer to the soil pellet and repeat the
homogenization and differential centrifugation steps two more times (i.e.,
repeat steps 2-4 combining the bacterial pellets in step 4).
6. Wash the cells by carefully resuspending the cell pellet in 200 ml of TE using
a small, clean paint brush. Collect the bacteria by centrifugation in a Sorvall
GSA rotor at 12,000 rpm (14,740 x g) for 20 min at 4° C.
f
1. Gently resuspend the cell pellet in 20.0 ml of TE (again using a paint brush),
transfer the cell suspension to a 50 ml Oak Ridge tube, then add 5.0 ml of 5 M
NaCl and 125 uJ of 20% Sarkosyl and incubate at room temperature for 10
min.
8. Collect the cells by centrifugation in a Sorvall SS34 rotor at 12,000 rpm (11,220
x g) for 20 min at 4° C.
9. Gently resuspend the cell pellet in 3.5 ml of Tris/sucrose/EDTA with a paint
brush.
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27
10. Add 0.5 ml of lysozyme solution, mix by vortexing then incubate at 37° C for
30 min without shaking.
11. Add 0.5 ml of pronase E, mix by vortexing, then incubate at 37° C for 30 min
without shaking.
12. Transfer to a 65° C water bath for 10 min, then add 250 ul of 20% Sarkosyl and
incubate at 65° C for 40 min.
13. Transfer to ice and let stand for at least 30 min.
14. Clear the lysate of cellular debris by centrifugation in a Sorvall SS34 rotor at
18,000 rpm (25,260 x g) for 1 h at 4° C.
15. Carefully transfer the supernatant to a clean Oak Ridge tube and" add 9.0 ml
of sterile distilled H20, 12.7 g of finely ground cesium chloride and 1.5 ml of
ethidium bromide (10 mg/ml). Mix by gentle inversion until the cesium
chloride is dissolved and adjust the refractive index to 1.3865-1.3885 (these
values correspond to a density range of 1.55-1.58) by adding cesium chloride
(to increase the value) or distilled H20 (to decrease the value):
16. Transfer the mixture to an ultracentrifuge tube, fill the remaining volume
and balance the tubes using cesium chloride balance solution, then seal the
tubes and band the DNA by ultracentrifugation in a Sorvall TV865B rotor at
52,000 rpm (255,800 x g) for 9-16 h at 18° C.
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28
17. Fractionate the DNA band with a 5 ml syringe and needle (this procedure is
detailed below in the section entitled "Fractionation of DNA Bands from
Cesium Chloride Gradients"), transfer the solution to a clean ultracentrifuge
tube, fill the remainder of the tube with cesium chloride balance solution and
repeat the ultracentrifugation step. This second round of ultracentrifugation
results in a substantial increase in purity of the DNA obtained by diluting the
contaminants (which are dispersed throughout the gradient) without
diluting the DNA (which forms a discrete band in a small volume).
18. Fractionate the DNA band and process through isopropanol extraction,
desalting and concentrating DNA by ethanol precipitation, and DNA
quantitation as described in the appropriate sections below.
n. Direct Lysis
Materials
NOTE: The materials list is based on the simultaneous processing of eight 10 g
soil samples from the stage of sieved soil to the point of the final equilibrium
gradient centrifugation step. Larger numbers of samples can be accomodated by
either staging the ultracentrifuge runs, or by scaling the protocol to allow the use
of other rotors such as the Sorvall T1270 rotor which accommodates twelve 12.5 ml
samples, or the Sorvall TFT45.6 rotor which accommodates forty 6.0 ml samples.
This list is based on the items used in this laboratory and does not constitute a
commercial endorsement of any supplies or equipment. Reasonable substitutions
for particular types of centrifuge rotors, tubes and other materials and equipment
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29
that maintain, or reasonably approximate, the specified conditions are
appropriate.
1. Twenty-four 50 ml polycarbonate Oak Ridge tubes
2. Vortex mixer
3. Water bath at 70° C
4. Glass beads: two sizes are used; 0.7-1.0 mm (Sigma #G9393) and 0.2-0.3 mm
(Sigma #G9143)
5. Reciprocal platform shaker
6. Sorvall superspeed centrifuge (e.g., model RC5B) with an SS34 (8-place) rotor
or an SAGOO (12-place) rotor
7. Refractometer (to measure the initial density of the cesium chloride gradient
for DNA purification. If a refractometer is not available, density can be
determined by measuring the specific gravity of the solution).
8. Sorvall ultracentrifuge with TV865B rotor
9. Sixteen ultracentrifuge tubes
Recipes
NOTE: The recipes presented here are for reagents required from the stage of
sieved soil to the point of the final equilibrium gradient centrifugation step. After
this point the steps are common to both DNA isolation protocols and can be found
in the section titled: "Fractionation of DNA Gradients, Final Purification and
Quantitation of Bacterial Community DNA".
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30
1. Sodium Phosphate buffer (1 mM at pH 7.0)
Buffers the soil suspension during the lysis of bacterial cells.
•This solution can be made by combining 2.1 ml of 0,2 M NaH2PO4 and 3.3
ml of 0.2 M Na2HP04 in a total of 11 of distilled H20.
2. Cesium chloride balance solution (Rf = 1.3870)
This solution is used to bring cesium chloride gradient tubes to final
volume and to balance the tubes prior to ultracentrifugation.
for approximately 300 ml:
•Add 250 g of finely ground cesium chloride (CsCl) to 250 ml of distilled
H20 (sterile) and mix by inversion until the CsCl is dissolved.
"Add 12.5 ml of 10 mg/ml ethidium bromide.
•Check the refractive index and adjust as necessary to achieve an Rf of
1.3870 (this corresponds to a density of 1.56) by adding CsCl to increase
the refractive index (density) or H^O to decrease the refractive index.
NOTE: Refractive index is a measure of the density of the solution. In
lieu of using a refractometer, the investigator can determine the specific
gravity of the solution and adjust the density of the solution to 1.56.
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31
Procedure
NOTE: This protocol describes the steps involved from the stage of sieved soil to the
point of the final equilibrium gradient centrifugation step. After this point the
steps are common to both DNA isolation protocols and can be found in the section
titled: "Fractionation of DNA Gradients, Final Purification and Quantitation of
Bacterial Community DNA".
1. Add 20 ml of NaPC>4(1.0 mM, pH 7.0) and 0.25 g of sodium dodecyl sulfate
(SDS) to each 10 g soil sample in a 50 ml Oak Ridge tube, mix by vortexing
until thoroughly suspended, then incubate for 30 min at 70° C mixing every 5
minutes.
2. Add 5 g of large glass beads (0.7-1.0 mm) and 5 g of small glass beads (0.2-0.3
mm) and shake for 30 min by placing horizontally on a reciprocal platform
shaker at high speed (-100 oscillations/min) at room temperature.
3. Pellet soil and cell debris by centrifugation in a Sorvall SS34 rotor at 10,000
rpm (7,796 x g) for 10 min at 10° C.
4. Transfer the supernatant to a clean Oak Ridge tube and incubate on ice. for
15-30 min to precipitate the SDS. Clear the lysate by centrifugation in a
Sorvall SS34 rotor at 10,000 rpm (7,796 x g) for 10 min at 10° C, then carefully
transfer the cleared lysate to a clean Oak Ridge tube.
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32
5. Adjust the volume of the lysate to 15.5 ml with distilled H20, then add 14.5 g
of finely ground cesium chloride. Mix by gentle inversion until the cesium
chJoride is totally dissolved, then let stand at room temperature for 10-15 min
to precipitate proteins. Clear the lysate by centrifugation at 5,000 rpm (1,949 x
g) for 10 min at 10° C. The precipitated proteins will form a floating layer
that may appear "foamy"; this layer should be discarded.
6. Transfer the mixture to an ultracentrifuge tube containing 0.65 ml of
ethidium bromide (10 mg/ml) and mix by gentle inversion. Fill the
remainder of the tube with cesium chloride balance solution (Rf = 1.3870),
then balance the tubes, seal and band DNA by centrifugation in a Sorvall
TV865B rotor at 52,000 rpm (255,800 x g) at 18° C for 9-16 h.
7. Fractionate the DNA band with a 5 ml syringe and needle (this procedure is
detailed below in the section entitled "Fractionation of DNA Bands from
Cesium Chloride Gradients"), transfer the solution to a clean ultracentrifuge
tube, fill the remainder of the tube with cesium chloride balance solution and
repeat the ultracentrifugation step. This second round of ultracentrifugation
results in a substantial increase in purity of the DNA obtained by diluting the
contaminants (which are dispersed throughout the gradient) without
diluting the DNA (which forms a discrete band in a small volume).
8. Fractionate the DNA band and process through isopropanol extraction,
desalting and concentrating DNA by ethanol precipitation, and DNA
quantitation as described in the appropriate sections below.
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33
IE. Fractionation of DNA Gradients, Final Purification
and Quantitation of Bacterial Community DNA
NOTE: At this stage, the total bacterial community DNA has been extracted from
soil and is in aqueous solution. Most of the subsequent protocols and analyses are
routine molecular biology protocols with some notable differences described
elsewhere in this volume. For further information on routine techniques of
molecular biology and/or as an additional resource, the reader is referred to the
many molecular biology and cloning laboratory manuals that are available (e.g.,
Ausubel et al., 1990; Sambrook et al, 1989).
ITJ a. Fraetionation of DNA Bands From Cesium Chloride Gradients
Materials
1. Syringes (5 ml) fitted with 18 gauge needles
2. Hand-held ultraviolet light source (e.g., Blak-ray model B-100A, VWR
Scientific Co.) with UV safety glasses or goggles for eye protection
Procedure
L Stop the ultracentrifuge and carefully remove the tubes from the rotor. Avoid
shaking the tubes as this will perturb the gradients. It is best to let the rotor
coast to a stop from 3,000 rpm to zero rather than using the brake which may
cause some loss of resolution of the DNA band in the gradient.
-------
2, Extract DNA bands in 1-2 ml volume under ultraviolet illumination using a 5
ml syringe and 18 gauge needle as follows:
•Poke a hole at the very top of the ultracentrifuge tube to allow air in.
•Insert the needle and syringe just below the visible DNA band with the
needle orifice pointed up and extract the DNA band by slowly
withdrawing the plunger of the syringe,
NOTE: Wear gloves and UV goggles or glasses to protect yourself from
the ethidium bromide and UV irradiation.
3. Proceed to isopropanol extraction of ethidium bromide as described below or
prepare for second banding as described above.
EQ b. Isopropanol Extraction of Ethidium Bromide From DNA
Materials
1. Six ml Falcon tubes (capped polypropylene tubes)
2. Thirty ml Corex tubes (glass centrifuge tubes)
3. Sorvall superspeed centrifuge with an SS34 (8-place) or an SA600 (12-place)
rotor
4. Capacity for drying under vacuum (e.g., lyopholizer)
NOTE: air drying can be substituted for drying under vacuum
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35
Recipes
1, Isopropanol saturated with 5 M sodium chloride
This is used to remove ethidium bromide from DNA following the
ultracentrifugation steps,
for approximately 1 1:
•Prepare 1 1 of 5 M sodium chloride (NaCl) in a 2 1 bottle and autoclave.
•After the 5 M NaCl cools, add 1 1 of isopropanol to the bottle, mix
thoroughly and let sit until the organic and aqueous phases separate.
There will be some volume loss as the water is mixed with the alcohol.
•Add additional isopropanol as necessary until there is some NaCl
precipitate present after the phases separate.
Procedure
1. Fractionate the DNA band (in 1-2 ml volume) as described in the previous
section and transfer to a 6.0 ml Falcon tube.
2. Add an equal volume of isopropanol saturated with 5 M NaCl.
3. Mix by gentle inversion and let sit until the phases separate.
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36
4, Pipette oft the top layer (isopropanol) using a Pasteur pipet and discard in an
appropriate fashion (the isopropanol will be pink in color and contains
ethidium bromide).
5. Repeat steps 2-4 until all pink color is gone and then once more (usually a
total of 5 extractions). Proceed to desalting and concentration of DNA as
described below.
HI c. Desalting and Concentration of DNA
Materials
1. Pipetman or equivalent pipettors for small volumes
2. Eppendorf or equivalent microfuge and 1.5 ml microfuge tubes
Recipes
1. 3 M Sodium Acetate (NaOAc), pH 5.2
Monovalent cations must be present when precipitating DNA in the
presence of alcohol to provide for quantitative recovery of DNA,
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37
for 250 ml:
•Dissolve 61.52 g of anhydrous NaOAc in 150 ml of distilled H20.
•Adjust the pH to 5.2 with glacial acetic acid.
•Adjust the volume to 250 ml with distilled H2O, then autoclave.
Procedure
1. Following the removal of ethidium bromide transfer the DNA solution to a
labelled Corex tube, add two volumes of sterile distilled H20, then add 2
volumes of cold (-20° C) 100% ethanol, cover with parafilm and mix
thoroughly by inversion or vortexing.
e.g., DNA volume 1.5 ml
2 volumes distilled H20 3.0ml
2 volumes (6 x original DNA volume) ethanol 9.0 ml
Incubate overnight at -20° C.
NOTE: This first precipitation should not be incubated more than 24 h or the
cesium chloride may crystallize out complicating further purification.
2. Pellet the DNA by centrifugation in a Sorvall SS34 (or SAGOO) rotor at 7,500
rpm (4,385 x g) at 4° C for 1 h. Position the tube so that label is toward the
outside of the rotor as a reference for the location of the DNA pellet which
may not be readily visualized at this stage.
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38
3. Discard (pour off) supernatant being careful not to disturb the pellet. Invert
the tube on a paper towel and drain dry (5 min),
4. Complete drying under vacuum. Complete air drying may be substituted for
vacuum drying.
5. Add 400 ul of sterile distilled H2O, mix by vortexing to dissolve the DNA (it
may be helpful to use a Pipetman P1000 to aid in resuspending the DNA
pellet by pipetting the 400 ^1 of distilled H20 up and down along sides of tube).
6. Transfer the DNA solution to a labelled 1.5 ml Eppendorf tube.
7. Collect the remaining liquid in the Corex tube by brief centrifugation and
transfer to the Eppendorf tube.
8. Add 40 til of 3 M sodium acetate (pH 5.2) and 880 ul of cold (-20° C) ethanol to
the tube, mix thoroughly by vortexing and incubate at -20° C for at least I h
(DNA may be stored for extended periods in 70% ethanol in the presence of
monovalent cations).
9, Collect the DNA by centrifugation in the microfuge for 15-30 min at 4° C.
10. Remove the supernatant with a Pasteur pipette and discard, wash the DNA
pellet once with cold (-20° C) 70% ethanol by gentle inversion, centrifuge
briefly (2 min), remove the supernatant with a Pasteur pipette and discard,
then dry under vacuum. Resuspend in a small volume (usually 100 |il).
Proceed to quantitation of DNA as described below.
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39
in dL Spectrophotometric Quantitation of DNA
Materials
1. Speetrophotometer with UV capabilities and quartz cuvette(s)
Procedure
The concentration of DNA in the sample can be measured by monitoring the
absorbance of a dilute solution of the sample at 260 and 280 nm as follows:
1. Dilute 5 ul of the DNA sample with 995 ul of distilled
2. Measure the absorbance of this solution at 260 and 280 nm.
3, Calculate the concentration of DNA in the sample based on the value of 1.0
Aaeo unit = 50 jig/ml of DNA, and taking into account the 1:200 dilution factor
of the sample.
4. Calculate the ^260/^280 ra^io- This ratio indicates the degree of
contamination of the DNA with humic (phenolic) compounds and proteins
since these molecules exhibit strong absorbance at 280 nm. Pure DNA has a
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40
A260/A280 rat»° of 2,0 with a value of 1.7-2.0 indicating relatively pure DNA.
DNA solutions can be stored at 4° C but, preferably, at -20° C.
NOTE: As mentioned previously, the precise quantitation of bacterial DNA
isolated from the soil environment can be problematic due to the copurification of
humic contaminants which also absorb light in the UV range. If there is a
distinct brownish tinge to the DNA solution, or if the ^2B(^^28Q ratio is low this
indicates that there is significant contamination of the DNA with compounds
from the soil. If such contaminants are present and precise quantitation is
desirable, alternate methods of quantitation must be employed. Perhaps the
simplest way to precisely quantitate DNA in the presence of humic contaminants
is to measure the UV fluorescence of the DNA in the presence of ethidium
bromide. This is readily accomplished using agarose gel electrophoresis by the
protocol described in the next section.
IH e. Quantitation of DNA by UV Fluorescence in the Presence of
Ethidium Bromide
NOTE; For this analysis a relatively high agarose concentration of 1.2% is used.
This allows the contaminants to migrate significantly far into the gel while the
randomly sheared (but generally large) DNA fragments migrate as essentially a
single band near the origin.
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41
Materials
1. Submarine agarose gel apparatus. There are a wide variety of these
available commercially. It is appropriate to obtain one that will also be useful
for subsequent analyses of the bacterial community DNA. A gel with
minimum dimensions of 15 x 15 cm is recommended to allow sufficient
resolution of differently sized DNA fragments and longer gels (e.g., 15 x 25
cm) might be more useful since more than one set of wells can be cast into a
single gel as is desirable for this analysis.
2. Power supply for agarose gel electrophoresis
3. Microwave oven or heated stirring plate
4. Access to a camera set-up suitable for photographing gels under ultraviolet
illumination such as a transilluminator/Polaroid camera set-up which is
basic equipment in most molecular biology laboratories
5. Access to equipment for densitometric analysis of the gel photograph is
desirable, as it will allow more precise quantitation, but not essential.
Recipes
1. TAB (Tris-acetate-EDTA) Buffer
This buffer has appropriate characteristics of pH, buffering capacity and
ionic strength which will result in clearly resolved DNA bands in an
agarose gel.
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42
Contains:
40 mM Tris-acetate
1 mM EDTA
for 1 1 of 20 X stock:
•Combine the following in 800 ml of distilled H20:
96.8 g Tris-base
22.84 ml glacial acetic acid
40 ml 0.5 M EDTA (pH 8.0)
Adjust to pH 8.0, if necessary, using glacial acetic acid, bring to 1 1 final
volume with distilled H20
2. 5 X sample loading dye (for agarose gels)
This dye allows tracking of the progress of electrophoresis by monitoring
the dye front and also makes the DNA samples sufficiently dense to sink to
the bottom of the wells in the submarine gel.
Contains:
100 mM EDTA
50% glycerol
0.15% bromophenol blue
0.15% xylene cyanole
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43
for 10 ml;
•Combine the following:
2 ml of 0.5 M EDTA, pH 8.0
5.0 ml of 100% glycerol
0.75 ml of 2% bromophenol blue
0.75 ml of 2% xylene cyanole
1.5 ml of distilled H20
Procedure
1. Combine an appropriate volume of 1 X TAB buffer with an appropriate
amount of agarose in an Ehrlenmeyer flask (these values are determined
based on the gel dimensions and the desired percentage of agarose; 1.2% for
this analysis).
2. Swirl the flask to evenly distribute the agarose.
3. Heat the solution until the agarose is completely dissolved; undissolved
agarose will appear as flecks in an otherwise clear solution. If using a
microwave, heat at high power for 2 min or until the mixture bubbles.
Remove the flask from oven (before it boils over), carefully swirl again, and
reheat until all of the agarose goes into solution.
4. Place the flask containing the molten agarose in a 55-65° C water bath or on
the benchtop to cool. The gel should be poured when the temperature of the
solution is 55-65° C (almost too hot to hold).
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44
5, Prepare the gel apparatus for casting the gel while the agarose is cooling.
There are several different types of gel boxes and these preparations will
depend on the particular one you are using.
6. Just prior to pouring the gel, add ethidium bromide to a final concentration
of 0.5 ug/ml to the dissolved agarose and swirl to mix (for agarose gels it is
convenient to have a 1 mg/ml stock solution of ethidium bromide which can
be made by dilution from the 10 mg/ml stock described above).
7. Pour the agarose solution into the gel casting tray and adjust the well-
forming comb(s) to keep the wells properly aligned. Allow the agarose to cool
and solidify (-20-30 min) prior to use.
8. To prepare gel for running:
•Fill the electrophoresis tank (apparatus) with buffer solution (IX TAB
containing 0.5 (ig/mJ ethidium bromide) and place the gel (still in the
casting tray) on the tank platform. The buffer must cover the gel by 1-2
mm.
•Carefully (to avoid breaking the walls of the wells) remove the comb.
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45
9. To prepare samples for loading:
•In a microfuge tube bring ~1 ug of total bacterial community DNA
solution (as determined spectrophotometrically) to a total volume of 20 ul
with distilled H20. Add 5.0 ul of 5 X sample dye to the sample and mix.
•A set of DNA standards should be prepared by serial dilution of a known
amount of ultrapure DNA (an appropriate source would be to purchase
undigested phage lambda DNA commercially). The dilution range
should be from 3.0 ug to 0.1 ug in 20 ul volume since the concentration of
humic-contaminated DNA is generally overestimated. Add 5.0 ul of 5 X
sample dye to each sample and mix.
10. Load the samples and standards into the gel wells using the Pipetman
pipettor. Stick the tip below the surface of the buffer but above the well
bottom, and dispense the sample slowly. The sample will sink through the
buffer and settle in the well. It is recommended that the DNA samples and
standards be run near each other in the same gel for more precise
quantitation. An appropriate way to accomplish this is to cast two sets of
wells into the gel; the upper one (nearer the origin) for the DNA standard
dilution series and the lower for the DNA samples of unknown
concentration.
11. After the gel has been loaded, gently place the cover on the apparatus and
hook up the power leads. DNA is negatively charged and will migrate
toward the positive (red lead and jack in power supply) electrode. Adjust the
power to 50 volts (constant voltage). Run the gel until the leading dye front
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46
(bromophenol blue) has migrated two-thirds the length of the gel or two-
thirds of the way to the second set of wells.
12. Photograph the gel under ultraviolet illumination either from a
transilluminator or by even distribution of UV illumination from a hand-
held source.
13. Determine the concentration of the DNA in the original sample by
comparison to the known standards using a densitometer or by visual
examination. The DNA will fluoresce bright orange and be found relatively
near the origin of the gel. The humic contaminants, if significant amounts
are present, will fluoresce blue-green and should migrate well ahead of the
DNA in the agarose gel.
ACKNOWLEDGEMENTS
The author's work has been supported by U.S. Environmental
Protection Agency Agreement CR 814575 and National Science
Foundation Science and Technology Grant No. DIR 8809640.
Although the research described in this article has been funded
in part by the EPA, it has not been subjected to the Agency's
review and therefore does not necessarily reflect the views of
the Agency, and no official endorsement should be inferred.
The author gratefully acknowledges the technical support
provided by Bernard M. Schroeter and Robert A. Laymen.
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47
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_ TECHNICAL REPORT UATA
(ftcfftt ntd Imttruttions on tht nvtnt it/art eomptei
i. REPORT NO.
EPA/600/A-.93/013
2.
4. TITLE AND SUStlTLE
Isolation and purification of
bacterial DNA from soil
PATE
I. PERPORMINB ORBANIZATION CODE
7. AVTHOMS)
William E. Holben
, PERFORMING ORBANIEATiON REPORT NO.
ORGANIZATION NAMI AND ADDRUC
tO. PROORAM ELEMENT NO.
Michigan State Univ. , East
Lans ing , MI
Tl. CONTRACT/BRANT NO,
13. SPONSORING AGENC* NAMI AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97333
11. TVfi Of HtfJDftT AND PIMIOD COVERED
Book Chapter
i«. SPONSORING AOENCV CODE
EPA/600/02
16.SUFPLSMENTA*Y NOTEfS
1992 Chapter in: Methods of Soil Analysis
Society of America)
(pub. Agronomy
Recently, new methods for monitoring specific bacterial populations in
environmental samples have become available. These methods employ the
techniques of molecular biology to distinguish, enumerate and monitor
individual bacterial populations within a microbial community by the
detection of DNA sequences specific to those populations using
appropriate molecular probes. DNA-based detection of bacterial
populations helps to overcome a major limitation of microbial ecology
and soil microbiology; the difficult task of specifically monitoring an
individual population of microbes in the environment, and in the
presence of the entire microbial community. Such capabilities are
essential to understanding the complex interactions between the
environment, other microorganisms and the population(s) of interest^-—
DNA-based detection of microbial populations thus represents a new tool
to expand the capabilities of investigators to detect and quantify
microorganisms in environmental samples.
KEY WORDS AND DOCUMENT ANALYSIS
MsemrroRS
fc.!DENTiFtEMft/OPEN ENDED TERM* C. COfATI F*ld/Gl0up
DNA, monitoring, bacterial
populations,
I. DISTMlSI/TiON STATEMENT
Release to Public
.
nclassifie
It. NO. OF PACES
51
B SECURITY CLASS (TMtmttl
Unclassified
33. PRICE
•PA
uae-i c»-»i)
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