was originally developed for Listeria monocytogenes but has worked well
with other Gram+ bacteria
1. Pellet cells from 10 ml overnight cultures in BHI or LB and wash in
5 ml of 0.1X SSC.
2. Resuspend in 1 ml 0.01 NA-PHOSPHATE BUFFER (We have this buffer for
protein purification, come to get) in 20 % SUCROSE (pH 7.0), add lysozyme
to 2.5 mg/ml, and incubate at 37 C for 45 min or longer.
3. Add 9 ml LYSIS BUFFER (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 500 mg pronase
B, 1 % SDS), and incubate additional 30 min (longer) at 37°C.
4. Phenol and chloroform extract lysed cells, and ethanol precipitate
the DNA with 0.1 vol. 3 M sodium acetate, pH 4.8 and 2 vol. 95% ethanol.
5. Spool out DNA with a glass rod, wash once with 80% ethanol before drying.
(Or by centrifugation)
Some bacterial species may require a longer incubation in lysozyme. For
Renibacterium salmoninarum (a G+ salmon pathogen we work with), lysozyme
incubations overnight at 37 C worked very well with high yields of DNA
A. PHENOL/CHLOROFORM EXTRACTION OF DNA
· phenol:chloroform (1:1)
1. Add an equal volume of buffer-saturated phenol:chloroform (1:1) to
2. Mix well. Most DNA solutions can be vortexed for 10 sec except for
molecular weight DNA which should be gently rocked.
3. Spin in a microfuge for 3 min.
4. Carefully remove the aqueous layer to a new tube, being careful to
avoid the interface. (Steps 1-4 can be repeated until an interface is
no longer visible)
5. To remove traces of phenol, add an equal volume of chloroform to the
6. Spin in a microfuge for 3 min.
7. Remove aqueous layer to new tube.
8. Ethanol precipitate the DNA
Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample. Typically,
an equal volume of TE-saturated phenol is added to an aqueous DNA sample
in a microcentrifuge tube. The mixture is vigorously vortexed, and then
centrifuged to enact phase separation. The upper, aqueous layer carefully
is removed to a new tube, avoiding the phenol interface and then is subjected
to two ether extractions to remove residual phenol. An equal volume of
water-saturated ether is added to the tube, the mixture is vortexed, and
the tube is centrifuged to allow phase separation. The upper, ether layer
is removed and discarded, including phenol droplets at the interface.
After this extraction is repeated, the DNA is concentrated by ethanol
1. Add an equal volume of TE-saturated phenol to the DNA sample contained
in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.
2. Centrifuge the sample for 5 minutes at room temperature to separate
3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully
avoiding proteins at the aqueous:phenol interface. At this stage the aqueous
phase can be extracted a second time with an equal volume of 1:1 TE-saturated
phenol:chloroform, centrifuged and removed to a clean tube as above but
this additional extraction usually is not necessary if care is taken during
the first phenol extraction.
4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge
for 3 minutes at room temperature. Remove and discard the upper, ether
layer, taking care to remove phenol droplets at the ether:aqueous interface.
Repeat the ether extraction.
5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate,
as discussed below.
B. Concentration of DNA by ethanol precipitation
Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to
the DNA sample in a microcentrifuge tube, which is placed in an ice-water
bath for at least 10 minutes. Frequently, this precipitation is performed
by incubation at -20°C overnight. To recover the precipitated DNA,
the tube is centrifuged, the supernatant discarded, and the DNA pellet
is rinsed with a more dilute ethanol solution. After a second centrifugation,
the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.
1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample
contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate
in an ice-water bath for at least 10 minutes. It is possible to place
the sample at -20°C overnight at this stage.
2. Centrifuge at 12,000 rpm in a microcentrifuge (Fisher) for 15 minutes
at 4°C, decant the supernatant, and drain inverted on a paper towel.
3. Add 80% ethanol (corresponding to about two volume of the original
sample), incubate at room temperature for 5-10 minutes and centrifuge
again for 5 minutes, and decant and drain the tube, as above.
4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about
5-10 minutes, or until dry.
5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA
(termed 10:0.1 TE buffer).
6. It is advisable to aliquot the DNA purified in large scale isolations
(i.e. 100 mg or more) into several small
(0.5 ml) microcentrifuge tubes for frozen storage because repeated freezing
and thawing is not advisable.
Notes on precipitation of nucleic acids
A. General rules
Most nucleic acids may be precipitated by addition of monovalent cations
and two to three volumes of cold 95% ethanol, followed by incubation at
0 to -70 °C. The DNA or RNA then may be pelleted by centrifugation at
10 to 13,000 x g. for 15 minutes at 4°C. A subsequent wash with 70%
ethanol, followed by brief centrifugation, removes residual salt and moisture.
The general procedure for precipitating DNA
and RNA is:
1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution
to be precipitated,
2. Add two volumes of cold 95% ethanol,
3. Place at -70°C for at least 30 minutes, or at -20°C overnight.
1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc (pH 4.8) and 1ml
of sterile water. Mix by inversion and store at -20°C.
2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid
solution to be precipitated.
3. Place at at -70°C for at least 30 minutes or -20°C for two hours
* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc.
DNA also may be precipitated by addition of 0.6 volumes of isopropanol.
B. Notes on phenol extraction of nucleic acids
The standard and preferred way to remove proteins from nucleic acid solutions
is by extraction with neutralized phenol or phenol/chloroform. Generally,
samples are extracted by addition of one-half volume of neutralized (with
TE buffer, pH 7.5) phenol to the sample, followed by vigorous mixing for
a few seconds to form an emulsion. Following centrifugation for a few
minutes, the aqueous (top) phase containing the nucleic acid is recovered
and transferred to a clean tube. Residual phenol then is removed by extraction
with an equal volume of water-saturated diethyl ether. Following centrifugation
to separate the phases, the ether (upper) phase is discarded and the nucleic
acid is ethanol precipitated as described above.
A 1:1 mixture of phenol and chloroform also is useful for the removal
of protein from nucleic acid samples. Following extraction with phenol/chloroform,
the sample should be extracted once with an equal volume of chloroform,
and ethanol precipitated as described above.