Molecular Biology Protocols
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" = microliter
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, etc.
"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 |
Solution II |
Solution III |
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 |
References:
Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation
of plasmid DNA. Methods Enzymol. 100, 243 - 255.
Biologie Moléculaire: encore des protocoles
de minipreps
sommaire