Avoiding phenol-chloroform extraction - I want to save time! (Oct/02/2005 )
Is anyone experienced with restriction digests of plasmids recovered using the alkaline method without performing phenol-chloroform extraction? I mean, after doing the miniprep, can I just precipitate with ethanol and do a digestion?
We avoid phenol-chloroform by using a column-based miniprep kit (we use the one from Qiagen, but they're all pretty much the same).
Depending on what you're going to use the DNA for, I suppose you could get away with precipitating from the cleared lysate...
I haven't read these classic papers in a while, but you might want to take a look and see if they tried precipitating right from the cleared lysate:
Birnboin H. C., and Doly J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513−1523.
Kado C. I., and Liu S-T. 1981. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365-73.
I'd also have a look at Biotechniques; they're usually full of articles dealing with rapid plasmid purification.
It usually depends of your host bacteria some of them like DH5alpha don't produce endonuclease so if you prepare DNA with a kick boiling method it might gave you digestable DNA !
I have done this many times without the phenol chloroform on plamsid DNA isolated from DH5a. Like the other post said, it could change with different strains, but this has worked for me.
I too have done this a million times and it works fine with JM107 and JM109 (just pay attention to the methylation status for some enzymes, like you would anyways)
I miniprep with a homebrew alkaline lysis, then Etoh / acetate ppt, Etoh wash, then proceed to digestion and ligation
Thank you all for your answers!