This was probably derived from Birnboim and I've used
it since 1984 for isolation of recombinant plasmids from E. coli
for routine screening. All solutions are listed at the bottom. "ul"
From overnight cultures (LB, BHI, other E. coli broths):
- Pour 1.5 ml into 1.5 ml microfuge tubes.
- Centrifuge 1 min in microfuge and carefully aspirate off the medium.
- Add 100 ul of Solution I and resuspend by vortexing.
- Add 200 ul of Solution II and mix completely by inversion. The cells
should lyse and turn somewhat clear and viscous.
- Let stand ~3 min and then add 150 ul of Solution III. Mix again by
inversion--a white clot of DNA/protein/SDS should form. Incubate on
ice 10-30 min.
- Centrifuge for 5 min in microfuge.
- Pour off the supernatant (~400 ul) into a fresh 1.5 ml microfuge tube.
Add 1 ml 95% ethanol and mix well.
- Centrifuge in microfuge ~15 min. Carefully pour off ethanol and wash
pellet with 0.5 ml 80% ethanol. Either dry in speed vac or dessicate
with a wash of 95% ethanol and air dry.
- Resuspend pellet in 100-300 ul water or TE (volume dependent on copy
number of plasmid). Ready for restriction digestion, PCR, subcloning,
"ul" = microliter
These preps contain RNA so if this is a problem a standard RNAse treatment
will suffice. Further cleanup can be accomplished with phenol/chloroform
extractions but for routine screening and subcloning I never bother. Your
mileage may vary.
| Solution I
| 5 mM sucrose
10 mM EDTA
25 mM Tris, pH 8.0
| 0.2 N NaOH
1% (w/v) SDS
| 3 M sodium acetate, pH 4.8
Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation
of plasmid DNA. Methods Enzymol. 100, 243 - 255 [Abstract].