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EXPERIMENT 19
Isolation of Bacterial DNA
Theory
To isolate a functional macromolecular component
from bacterial cells, you must accomplish three
things. First, you must efficiently disrupt the bac-
terial cell wall and cell-membrane system to facili-
tate extraction of desired components. Second, you
must work under conditions that either inhibit or
destroy the many degradative enzymes (nucleases,
proteases) released during cell disruption. Finally,
you must employ a fractionation procedure that
separates the desired macromolecule from other
cellular components in satisfactory yield and purity.
The isolation of bacterial DNA described in this
experiment, patterned after the work of Marmur
(1961), accomplishes these objectives. Bacterial
cells are disrupted by initial treatment with the en-
zyme, egg-white lysozyme, which hydrolyzes the
peptidoglycan that makes up the structural skele-
ton of the bacterial cell wall. The resultant cell walls
are unable to withstand osmotic shock. Thus, the
bacteria lyse in the hypotonic environment. The de-
tergent, sodium dodecyl sulfate, (SDS, sodium do-
decyl sulfate) then completes lysis by disrupting
residual bacterial membranes. SDS also reduces
harmful enzymatic activities (nucleases) by its abil-
ity to denature proteins. The chelating agents, cit-
rate and EDTA (ethylenediamine tetraacetic acid),
also inhibit nucleases by removing divalent cations
required for nuclease activity.
This experiment employs a variety of fraction-
ation methods to purify the bacterial DNA. Per-
chlorate ion is used to dissociate proteins from
DNA. Chloroformisoamyl alcohol is used to de-
nature and precipitate proteins by lowering the di-
electric constant of the aqueous medium. The pre-
cipitated material is removed by centrifugation.
DNA is then isolated from the solution by ethanol
precipitation, which allows spooling of the long, fi-
brous DNA strands onto a glass rod. Although the
residual RNA and proteins in the solution also co-
agulate, they do not form fibers and are left in the
solution as a granular precipitate. The resultant
crude DNA is dissolved and precipitated again with
isopropanol under conditions that favor specific
DNA precipitation. These steps form a general pro-
cedure for the isolation of chromosomal DNA from
virtually any bacterium. If you intend subsequently
to assay the biological activity of the isolated DNA
in a transformation assay (as in Experiment 20), it
is necessary to start this isolation with the appro-
priate species and strain of bacteria possessing the
genotype of interest.
This experiment will also provide you with ex-
perience in characterizing DNA samples by deter-
mining their ultraviolet absorption spectra and ob-
serving their thermal denaturation by means of the
hyperchromic effect. These topics were discussed in
the introduction to Section V.
Supplies and Reagents
For growth of wild-type (Trp!) Bacillus subtilis (if this ex-
periment is to be done together with Experiment
20. Other kinds of frozen bacterial cells can be used
if Experiment 20 will not be performed.)
Stock culture of wild-type B. subtilis (Bacillus Genetic
Stock Center strain 1A2)
Inoculating needle
333
334 SECTION V Nucleic Acids
Luria broth (see Appendix)
37C Incubator-shaker
Fermenter
Centrifuges, continuous and laboratory
Sterile saline solution (8.5 g NaCl/liter)
0.15 M NaCl, 0.l M Na2EDTA
Lysozyme solution, 10 mg/ml
25% (wt/vol) SDS solution
5.0 M NaClO4
Chloroformisoamyl alcohol (24:1, vol/vol)
95% Ethanol
Dilute (1/10X) saline-citrate (0.015 M NaCl, 0.0015 M
Na3-citrate)
Standard (1X) saline-citrate (0.15 M NaCl, 0.015 M Na3-
citrate)
Concentrated (10X) saline-citrate (1.5 M NaCl, 0.15 M
Na3-citrate)
3.0 M Sodium acetate, 1 mM EDTA, pH 7.0
Isopropanol
Chloroform
Spectrophotometer and quartz cuvettes
Protocol
Growth of Wild-Type Bacillus subtilis for
DNA Isolation
This portion of the experiment may be performed
by an instructor in advance.
1. Inoculate (sterile technique) 100 ml of sterile
Luria broth (200- to 400-ml flask) with a sam-
ple of wild-type Bacillus subtilis obtained from
a stock culture slant. Incubate this solution on
a shaker at 37C for 15 to 24 hr (until the cul-
ture is densely turbid and is entering the sta-
tionary phase of growth).
2. Transfer the starter culture (sterile technique)
into a fermenter containing 10 to 12 liters of
sterile Luria broth and continue the bacterial
growth at 37C while stirring (!400 rpm) and
aerating at 1 liter of air per liter of fluid per
minute. Monitor the growth periodically by de-
termining the turbidity of the culture (ab-
sorbance at 660 nm) during the 3- to 5-hr
growth until this culture just enters the sta-
tionary phase of growth.
3. Stop the growth by cutting off both air and ag-
itation. Use a continuous centrifuge (e.g.,
Sharples) to harvest the cells as quickly as pos-
sible.
4. Suspend the cell paste in an equal volume of
cold (1 to 4C) sterile saline solution. Complete
the cell-washing procedure by harvesting the
B. subtilis cells (10 min, 20,000 ! g, 1 to 3C)
with a high-speed centrifuge.
5. Decant the supernatant fluid from the cell pel-
let after centrifugation and store the washed
cells in Parafilm or waxed-paper-wrapped lots
at !20C until use (2-g lots are convenient).
The expected yield is 35 to 45 g of cells per 10
liters.
Isolation of Bacterial DNA
1. Suspend 2 g of bacterial cell paste in 25 ml of
0.15 M NaCl, 0.1 M Na2EDTA solution. The
bacterial paste is most easily suspended by first
adding a small amount of the NaClEDTA so-
lution, making a slurry, and then slowly adding
the rest of the NaClEDTA solution. After
suspension, add 1 ml of a 10-mg/ml lysozyme
solution and incubate the suspension at 37C
for 30 min, gently agitating occasionally.
2. After the 30-min incubation, complete the ly-
sis of the bacteria by adding 2 ml of a 25% SDS
solution and heating this preparation for 10
min in a 60C water bath. Cool the solution to
room temperature in a bath of tap water.
3. Add 7.5 ml of 5.0 M NaClO4 to the 25 ml of
lysed bacteria cells and mix gently. Then add
32.5 ml of CHCl3 isoamyl alcohol (24:1) (i.e.,
a volume equal to the volume of lysed cell
preparation containing 1.0 M perchlorate).
Slowly shake the solution (3060 oscillations
per minute, by hand or on a low-speed shaker)
in a tightly stoppered flask for 30 min at room
temperature.
4. Transfer the suspension to a 250-ml polypropy-
lene centrifuge bottle, and separate the result-
ing emulsion by centrifuging for 5 min at
10,000 ! g at room temperature.
5. During the centrifugation step, remove any
residual enzymes from a glass rod by heating
one end of the rod in a flame and allowing the
sterile end to air cool in a thoroughly washed
250-ml beaker.
6. After the centrifugation step, carefully pipette
the clear aqueous phase (top layer) away from
the coagulated protein emulsion at the inter-
face between the aqueous and organic phases
of the centrifuged solution. Do not attempt to

remove all of the aqueous phase; rather, avoid
contaminating the aqueous phase with mater-
ial from the other layers. Place the aqueous
phase, which contains the extracted nucleic
acids, in the 250-ml beaker. Dispose of the
CHCl3-containing waste in the special container
provided by your instructor.
7. Gently stir the nucleic acid solution with the
sterilized rod while slowly and gently adding 2
volumes (about 60 ml) of 95% ethanol. Pour
the ethanol gently down the side of the beaker so
that it is layered over the viscous aqueous phase.
Continue to stir this preparation gently with the
sterilized glass rod so that the ethanol is very
gradually mixed throughout the entire aqueous
phase. Avoid stirring vigorously, which would
shear the DNA strands and make the DNA
more difficult to isolate. The DNA will form
a white fibrous film at the waterethanol in-
terface, and you should be able to spool all of
the gelatinous, threadlike, DNA-rich precipi-
tate onto the glass rod. Continue gentle stir-
ring until the aqueous and ethanol phases are
completely mixed and no more DNA precipi-
tate can be collected on the glass rod. Drain off
excess fluid from the spooled crude DNA by press-
ing the rod against the walls of the beaker until no
further fluid can be squeezed from the spooled
preparation.
8. Dissolve the crude DNA by stirring the glass
rod with its spool of material in 9 ml of dilute
(1/10X) saline-citrate in a test tube. Difficulty
in dissolving the sample indicates failure to re-
move sufficient alcohol from the sample dur-
ing step 7. If such difficulty is encountered,
continue working the sample within the solu-
tion until you obtain as uniform a suspension
as possible.
9. To the DNA solution add 1 ml of 3.0 M sodium
acetate, 1 mM EDTA, pH 7.0 solution and
transfer the preparation to a 100-ml beaker.
Gently swirl the sample while slowly dripping
in 6.0 ml of isopropanol. If fibrous DNA is
readily apparent, collect the DNA threads by
stirring and spooling with a sterilized glass rod
as described in step 7. If a gel-like preparation
develops, add an extra 1.0 ml of isopropanol
and stir to spool the DNA onto the sterilized
glass rod. Remove excess fluid from the spooled
DNA by pressing the sample against the walls
of the beaker.
EXPERIMENT 19 Isolation of Bacterial DNA 335
10. Wash the sample (by submersion and brief
swirling of the DNA on the rod) in test tubes
containing, in order, 10 ml of 70% ethanol
(made by adding 2.6 ml H2O to 7.4 ml of 95%
ethanol), and then 10 ml of 95% ethanol.
11. If you plan to isolate the DNA only for use in
the genetic transformation outlined in Experi-
ment 20, or if a day or longer will elapse be-
fore you will characterize the DNA as described
below, store the DNA in a stoppered tube (2C)
as a spool submerged on the rod in 95%
ethanol. Dissolve the DNA as described in step
1 of the following section when you are ready
to use it.
Characterization of DNA
1. Remove the rod and the spooled DNA from
the 95% ethanol, blot away all obvious resid-
ual fluid with a clean piece of filter paper, and
then dissolve the DNA by stirring the glass rod,
with its spooled DNA, in a test tube contain-
ing 10 ml of dilute (1/10X) saline-citrate (ster-
ile solution if the DNA is to be used in Experiment
20). This solution can be stored at 2C.
Spectral Characterization of DNA
2. Dilute a 0.5 ml sample of the DNA solution
with 4.5 ml of standard (1X) saline-citrate and
determine the absorbance of this diluted sam-
ple at 260 nm against a dilute saline-citrate
blank containing no DNA. If the absorbance at
260 nm is greater than 1.0, dilute the sample
with a precisely known volume of standard (1X)
saline-citrate until you obtain an absorbance of
between 0.5 and 1.0.
3. Determine the absorbance of this solution at 5-
nm intervals between 240 and 300 nm, so as to
obtain an absorption spectrum for the isolated
DNA. (Remember that quartz cuvettes must be
used in this wavelength range.) Blank the spec-
trophotometer to read zero absorbance with 1X
saline-citrate at each wavelength. (If equipment
for the automated collection of this absorbance
spectrum is available, follow the instructions
for that instrument.) Include this absorption
spectrum in your report.
4. Calculate the A260!A280 ratio for your sample,
and compare it with the value expected for pure
DNA (2.0).
336 SECTION V Nucleic Acids
5. Assuming that a 1-mg/ml solution of native
DNA has an absorbance at 260 nm of 20 and
that all of your absorbance at 260 nm is due to
native DNA, calculate the concentration (in
milligrams per milliliter) of the DNA in your
undiluted DNA solution and the total yield (in
micrograms or milligrams) of DNA you ob-
tained.
6. Assuming 100% recovery of the cellular DNA
and assuming the wet bacterial cell paste was
80% water, calculate the percent of the dry
weight in the bacterial cells that is DNA. How
do your results agree with the DNA content of
a typical bacterium, which is about 3% of the
cells dry weight? If your results do not agree
with this value, discuss possible reasons for the
discrepancy.
Melting of DNA The Hyperchromic Effect 6.
1. If your laboratory is equipped with a spec-
trophotometer with a thermoprogrammer de-
signed for determination of melting curves of
nucleic acid samples (Gilford, Beckman Instru-
ments), determine a melting curve of a sample
of your DNA dissolved in the required volume
(usually 1 ml or less) of standard (1X) saline-
citrate so as to give an initial absorbance of
about 1 at 260 nm. Use the results from the
preceding section to decide how much you
need to dilute the stock DNA solution. Follow
the instructions provided by the instruments
manufacturer in programming the rate of tem-
perature increase, protecting the sample against
evaporation, etc.
2. If your laboratory does not have access to a
spectrophotometer with a thermoprogrammer,
use the quick cool method described in steps
3 through 6 below to obtain a melting curve
for your DNA. This method does not observe
a true equilibrium between native and dena-
tured (melted) DNA and is appreciably less
accurate than the method described in step 1,
but it will suffice to illustrate the principles un-
derlying the thermal separation of DNA
strands.
3. Using your stock DNA solution from step 11,
prepare 10 ml of a diluted DNA solution in the
dilute saline-citrate (1/10X) that has a final ab-
sorbance at 260 nm of about 0.5. Use the re-
sults from the preceding section to decide how
much you need to dilute the stock DNA solu-
tion. Determine the absorbance at 260 nm of
this dilute solution at room temperature (about
20C).
4. Transfer 1.0 ml of the same dilute DNA solu-
tion into each of six microcentrifuge tubes and
cap them tightly. Incubate one tube at each of
the following temperatures for 20 min: 50C,
65C, 75C, and 85C. In addition, incubate
two of the tubes at 100C (i.e., a boiling water
bath).
5. After 20 min of incubating the tubes at the
indicated temperatures, quickly cool all of
them by placing them in an ice-water bath and
gently agitating them. Allow one of the tubes
heated at 100C to cool slowly to room tempera-
ture by placing it in a rack at your bench (30 to
40 min will be required).
As quickly as possible after they have cooled,
determine the absorbance at 260 nm of the so-
lution in each tube. For a blank, use a sample
of dilute saline-citrate (1/10X ) that contains no
DNA.
7. During heating, the double-stranded structure
of DNA melts; that is, the strands separate.
The nucleotide bases in the separated strands
are no longer stacked in a regular fashion,
which results in an increase in their absorbance
of UV light. This is called the hyperchromic ef-
fect or hyperchromicity. When melted DNA is
quickly cooled in solutions with very low salt
concentrations, the strands are very slow to
reassociate into their original base-paired,
double-stranded form. Thus, you can estimate
the degree of melting at each temperature even
though you are measuring the absorbance at
260 nm at room temperature. The DNA sam-
ple that was heated to 100C and slowly cooled
to room temperature, on the other hand, is ex-
pected to form normal double-stranded DNA
again (reanneal) and to exhibit little hyper-
chromic effect. Do your data support this ex-
pectation?
8. Prepare a plot of the absorbance at 260 nm at
each temperature for the unheated sample and
each of the rapidly cooled samples. Estimate
the melting temperature (Tm) for your DNA.
(Tm is the temperature at which the DNA sam-
ple is half melted. Assume that the DNA is

completely melted at 100C. Calculate the hy-
perchromic effect for your DNA as the percent
increase in absorbance at 260 nm on complete
melting. Calculate the percent guanine plus cy-
tosine (% GC) base pairs in your DNA using
the following formula
% GC " 2.44 (Tm ! 81.5C ! 16.6 log10[Na!])
For dilute (1/10X) saline-citrate, this becomes
% GC " 2.44 (Tm ! 53.9C ). B. subtilis DNA
is known to contain about 43% GC. How does
this agree with your results? Discuss the results
of this experiment.
Exercises
1. The DNA of cells containing nuclei (eukary-
otic cells) is tightly associated with proteins
called histones, most of which are rather basic
proteins (i.e., they have high isoelectric points).
Considering the basic character of most his-
tones and the amino acids that contribute to
this basic character, what enzymes would prob-
ably be most effective in disrupting histones
from eukaryotic cellular DNA? Would you ex-
pect the procedure used in this experiment for
the isolation of bacterial DNA to be useful for
the isolation of DNA from eukaryotic cells?
2. This isolation of bacterial DNA utilizes
reagents in the neutral pH range. What effects,
if any, would you expect extremes in acid or al-
kali to have on the fibrous character of the
DNA isolated in this procedure? Why?
EXPERIMENT 19 Isolation of Bacterial DNA 337
3. Examine the procedure described in Experi-
ment 21 for the isolation of plasmid DNA from
bacteria. It is very different from the procedure
used to isolate chromosomal DNA in this ex-
periment. Could it be used to isolate chromo-
somal DNA from bacteria? Why or why not?
Explain why such different procedures are used
for isolating these two types of DNA.
4. What other physical and chemical treatments
besides heating would be expected to allow you
to demonstrate the hyperchromicity of single-
stranded DNA? Explain how these treatments
bring about strand separation.
5. Most naturally occurring RNA molecules are
singled-, not double-stranded duplexes, yet
RNA also shows a hyperchromic effect on
melting. Explain.
6. The equation used for calculating % GC bases
pairs in this experiment predicts that increas-
ing % GC and increasing Na! concentration
increases the Tm of DNA. Explain the physical
basis for these effects.
REFERENCES
Mandel, M., and Marmur, J. (1968). Use of ultraviolet
absorbance-temperature profiles for determining
the guanine plus cytosine content of DNA. Methods
Enzymol 12B:195.
Marmur, J. (1961). A procedure for the isolation of
desoxyribonucleic acid from microorganisms. J Mol
Biol 3:208.
Puglisi, J. D., and Tinoco, I. (1989). Absorbance melt-
ing curves of RNA. Methods Enzymol 180:304.