PLASMID
ISOLATION FROM BACTERIA
Introduction
In
virtually all bacterial species plasmids exist. These accessory genetic
elements typically account for only a small fraction of a bacterial genome
corresponding roughly to a range between 1 and 200 kb. Extremely large plasmids
with sizes far beyond 200 kb are also known. Plasmids of more than 50 kb might
be characterized as “large”, plasmids of less than 10 kb as “small”. The aim of
this compilation is to describe some fast methods for plasmid isolation leading
to “crude lysates”, the quality of which being sufficient for analytical
purposes, mainly agarose gel electrophoresis. By using few microliters of crude
lysates for agarose gel electrophoresis, the electrophoretic separation allows
conclusions on
* The presence
of plasmid DNA,
* The
determination of the molecular weight(s),
* The amount of
plasmid DNA due to the band intensity and on
* The purity of
the crude lysate.
The fast methods described here are often suitable for plasmid
screenings from bacteria in E. coli.
Principle
The procedures
are based on the fact that plasmids usually occur in the covalently closed
circular (supercoiled) ccc configuration within the host cells. After gentle
cell lysis all intracellular macromolecules have to be eliminated whereas
plasmid DNA is enriched and purified. The smaller a plasmid the easier is the
isolation of intact ccc molecules. DNA is very sensitive to mechanical stress,
therefore shearing forces caused by mixing/vortexing or fast pipetting must be
avoided as soon as cell lysis occurs. All mixing steps during and after cell
lysis should be performed carefully by inverting the tubes several times (8-10
fold). Especially in case of larger plasmids it is recommended to cut off the
ends of plastic pipette tips to minimize shearing forces. Gloves should be worn
in order to prevent contamination with DNases. Autoclaved (DNase-free) buffer
solutions, tubes and tips should be used. If phenotypic markers of a plasmid
(e.g. antibiotic resistances) are known, it is recommended to grow the cells
under selective pressure to avoid plasmid loss. If necessary, small plasmids of
Escherichia coli can easily be amplified using chloramphenicol. This results in
several thousand plasmid copies per cell leading to high DNA quantities
(Clewell, 1972). Large plasmids are maintained with only one copy per host
chromosome: visible DNA bands are more difficult to get.
For plasmid
isolation, bacterial cultures should be grown to late logarithmic/early
stationary phase. It is important to remove the supernatant completely after
centrifugation from the cell pellets. Tris buffer is the typical buffering
substance for DNA with buffering capacity in the slightly alkaline range in
which DNA can also be stored best (pH 7.5-8.2). EDTA is an important substance in plasmid
preparations because it inhibits nuclease activity. For long-term storage,
plasmid DNA should be frozen in aliquots of storage TE buffer. Repeated thawing
and freezing of DNA should be avoided.
Ethanol precipitation of
plasmid DNA
Measure the volume of the aqueous DNA
solution and mix gently with (10% v/v) 3 M Na-acetate, pH 5.2, then add double
of the total volume of pure ethanol (cooled to -20C), mix and leave for 10 min
in crushed ice. Spin for at least 30 min at room temperature. DNA precipitation
is not enhanced by long or low temperature incubation, whereas an extended centrifugation
time results in good DNA recovery.
RNase treatment
Prepare 100 ml of the following sterile TE buffer:
0.01 M Tris, pH 7.5, 0.001 M EDTA. Mix 1 mg of RNase A with 1 ml of this TE
buffer in an Eppendorf tube and incubate for 20 min in a boiling water bath to
eliminate DNases. Cool to room temperature, add the RNase solution to the
remaining 99 ml of the same TE buffer. This RNase buffer can be stored at 4°C
for a long time and is a good storage buffer for plasmid DNA. RNase is a very
stable enzyme and cleaves RNA within few minutes at room temperature.
Gel electrophoresis
Immediately before loading a gel, mix 8 μl of DNA
sample with 2 μl of loading buffer (0.05 M EDTA, 20% Ficoll, 0.25% bromophenol
blue, in H20).
When
using a horizontal electrophoresis apparatus (horizontal apparatus is the usual
and better type of electrophoresis), for quick analytical gels, mini-gels on
glass slides can be prepared as follows: about 25 ml of 0.8-1.0% low
electroendosmosis (EEO) agarose in TBE buffer (0.089 M Tris, 0.089 M boric
acid, 0.0025 M EDTA) are poured on a glass slide of approx. 10 x 7 cm.
Depending on the electrophoresis comb used, up to 14 samples can be run. The
same TBE buffer is used as electrophoresis buffer. Usually, the electrophoretic
separation is done at 30-90 V for 2-6 hours (to be tried out). For
visualization of DNA bands and photography, intercalating dyes like ethidium
bromide are used: After staining for 30-60 minutes in the dark, DNA bands can
be made visible under short wave length UV light.
Procedure:
1. Hot alkaline method for all plasmid
sizes and bacteria
Centrifuge 2-3 ml of culture, resuspend
pellet in 1 ml of solution containing 0.04 M Tris-acetate, pH 8.0 (adjust pH
with glacial acetic acid) and 2 mM EDTA
Add 2 ml of lysis buffer (0.05 M Tris,
3% SDS, pH 12.50, adjusted with 2 N NaOH) and mix
Incubate at 60-68°C for 30-45 min
(strain dependent)
Add to hot samples 6 ml of
phenol/chloroform (1:1) and mix gently to complete emulsification
Separate phases by centrifugation at
10.000 x g for 15-20 min at RT and transfer the upper aqueous phase carefully
(avoid interphase which contains debris) to new tube containing 1 volume of
chloroform. Mix and centrifuge again for separation of phases
Recover aqueous phase and use directly
for agarose gel
2. Lysozyme method for various
Gram-negative bacteria
Centrifuge 10 ml of culture, resuspend
pellet in 1.4 ml of the following TE buffer: 0.01 M Tris, pH 8.5 and 1 mM EDTA.
Transfer to Eppendorf tubes and spin for 3 min
Resuspend pellet in 0.4 ml of solution
(15% sucrose, 0.05 M Tris, pH 8.5, 0.05 M EDTA), mix vigorously, and cool on
ice
Add 0.1 ml of freshly prepared lysozyme
(5 mg/ml in TE buffer used above), mix carefully and incubate on ice for 20-40
min
Add 0.3 ml of pre cooled Triton buffer
(0.1% Triton X-100, 0.05 M Tris, pH
8.5, 0.05 M EDTA), incubate on ice for
20 min and centrifuge at 4°C for 4 min
Transfer clear supernatant into new tube
and add 4 μl of diethyloxydiformiate, mix gently
Incubate for 15 min at 70°C, cool for 15
min to RT, then incubate on ice for 15 min
Centrifuge for 4 min, transfer
supernatant into new tube, fill up with ¬20°C ethanol for DNA precipitation,
mix gently
Centrifuge for at least 30 min at RT,
dry pellet in vacuum dessiccator and resuspend in storage TE buffer or in RNase
buffer before use.
3. Lysis of cells from single colonies
on agarose gel
Transfer 1-2 freshly grown single
colonies with a toothpick into 20 μl of cold buffer (0.025 M Tris, pH 8.0, 25%
sucrose, 0.250 M EDTA, 7% Ficoll 400)
Add 20 μl of freshly prepared lysis
solution (0.1 mg/ml of lysozyme, 10 μl/ml of RNase A, in the above buffer), mix
well and immediately fill 10-15 of the mixture into the well of an agarose gel
which contains 0.5% SDS
Add as “upper layer” onto the cell
lysate 10 μl of the following solution: 0.025 M Tris, pH8.0, 10% SDS, 25%
sucrose, 0.07% bromophenol blue
After 15-30 min apply low voltage (half
of usual voltage) for 30 min, and then apply usual electrophoretical conditions
4. Isolation procedure for all plasmid
sizes from all bacteria
Centrifuge 2 ml of a culture and wash
pellet in 2 ml of the following TE buffer: 0.05 M Tris, pH 8.0, 0.01 M EDTA.
Resuspend in 40 μl of the same TE buffer
Fill 0.6 ml of freshly prepared lysis
buffer (TE buffer used above with 4%SDS, pH adjusted to 12.45) into Eppendorf
tube and add the cell suspension to the lysis buffer, mix gently
Complete lysis by incubating at 37°C for
20-30 min
Add 30 μl of 2 M Tris, pH 7.0 for
neutralization, mix gently
Add 024 ml of 5 M NaCl for precipitation
of chromosomal DNA and protein and incubate on ice for 4 hrs
Centrifuge for 10 min and transfer
supernatant into new tube for ethanol precipitation (as usual) or for previous
extraction with phenol/chloroform.
