In the following protocol, plasmid DNA is prepared from
small amounts of many different cultures of plasmid-containing
bacteria. Bacteria are lysed by treatment with a solution
containing sodium dodecyl sulfate (SDS) and NaOH (SDS denatures
bacterial proteins, and NaOH denatures chromosomal and plasmid
DNA). The mixture is neutralized with potassium acetate, causing
the plasmid DNA to reanneal rapidly. Most of the chromosomal DNA
and bacterial proteins precipitate -- as does the SDS, which
forms a complex with potassium -- and are removed by
centrifugation. The reannealed plasmid DNA is then concentrated
by ethanol precipitation. There are a million versions of this published
in Biotechniques. They all work more or less similarly. The
only variant we use is the "Alternate method" and has been
modified so that you can use the DNA for in vitro transcription
translation.
Minipreps of Plasmid DNA
Most manipulations are performed in microcentrifuge tubes.
This procedure is appropriate for preparation of small amounts of
DNA from 1 to 24 cultures of plasmid-containing bacteria.
Materials
LB medium
TE buffer
NaOH/SDS solution (Solution II)
Potassium acetate solution (Solution III)
100% ethanol
70% ethanol
20ug/ml DNase-free RNase (optional)
Inoculate 5 ml LB medium with a single bacterial colony.
Grow to saturation (overnight).
Spin 1.5 ml of cells 20 sec in a microcentrifuge to pellet.
Remove the supernatant with a Pasteur pipet, or aspirate
using
a pulled Pasteur pipet.
This spin can be at 4ºC or at room temperature
Resuspend pellet in 100ul glucose TE buffer (Solution
I).(Let sit 5 min at room temperature;optional)
Be sure cells are completely resuspended
Add 200ul NaOH/SDS solution (Solution II), mix by tapping
tube with finger,or inverting the tube gently twice,and
place on ice.
Add 150ul potassium acetate solution (Solution III) and
vortex at highest speed for 2 sec to mix.
Be sure mixing is complete
Spin 3 min in microcentrifuge to pellet cell debris and
chromosomal DNA.
This spin can be at 4ºC or at room temperature
Transfer supernatant to a fresh tube, phenol extract with
450ul of buffered phenol. Mix well, spin 5 seconds in
microfuge to separate phases. Remove organic phase (bottom)
carefully with a pulled Pasteur pipet and discard in
flammable waste disposal, not down the sink! Add 0.6 vol of
100% isopropanol (300ul), vortex to mix.
Spin 5-10 min at room temperature to pellet plasmid DNA and
RNA.
Remove supernatant, wash the pellet with 200ul of 70%
ethanol, and dry pellet under vacuum.
NOTE: ETHANOL
and ISOPROPANOL ARE HIGHLY FLAMMABLE; USE WITH CAUTION NEAR
OPEN
FLAME.
Resuspend the pellet in 20ul (TE buffer + 20ug/ml RNase).
1 to 2ul of the resuspended DNA should be used for a
restriction digest.
IMPORTANT NOTE: Phenol is an organic solvent and will cause
severe burns if it comes into contact with your skin! The smell
of it is also very unpleasant and irritating to the nostrils. It
should be used with great care, wearing lab coat, gloves, eye
protection and in the fume hood. Read the MSDS sheet before using
this material.
Troubleshooting
If no plasmid DNA is obtained, try leaving out the RNase,
isolating the plasmid DNA from a different strain, precipitating
the plasmid DNA with ammonium acetate, or extracting
with phenol. If the isolated DNA fails to cut with
restriction endonucleases, the most common cause is inadequate
washing of the pellets after the ethanol precipitation step.
Inadequate washing of the pellets is sometimes, but not always,
manifested in the pH of the final DNA solution suspended in TE
buffer, which should be >6.5. Placing 5ul of the prep onto a
piece of pH paper will establish this point. If the pH of the
DNA is normal but it still fails to cut, precipitating the DNA a
second time with ethanol, or washing the pellets from the first
precipitation with 70% ethanol, will usually clean up the DNA
enough to cut it. In general, pH problems can often be avoided
by aspirating off the supernatants from the isopropanol
precipitation and ethanol washes thoroughly.
Anticipated Results
Approximately 3ug DNA is obtained from 1.5 ml of a culture
of cells that contains a pBR322-derived plasmid. From a PUC based plasmid
you get 15-20 micrograms of DNA.
Time Considerations
With practice, this protocol can be used by a skilled
practitioner to produce 12 or 24 samples of DNA from 12 saturated
bacterial cultures in about 2.5 hr.
