Boiling Method for Minipreps

jchen at CALSHP.CALS.WISC.EDU jchen at CALSHP.CALS.WISC.EDU
Tue Aug 3 17:22:24 EST 1993


> 
> The boiling prep is wonderful, but don't even try it using HB101 as a host. 
> Re-transform your plasmids into DH5 alpha, DH5alpha F', DH1, even XL-1 Blue,
> but you will have absolutely no success with HB101 as a host:  DNA from this
> and many other commonly used hosts (like TG1) isolated by by boiling preps will
> degrade, due to a heat resistant endonuclease.  This is not just folklore; DH5
> alpha has an endonuclease-1 mutation that seems to make it perfect for boil
> preps. OK, you will get DNA from HB101 and it will probably even cut, but it
> will give you shitty sequence.  I've used DH5 alpha and Bluescript based
> vectors for plasmid sequencing since 1988, and in the hundreds of gels I've run
> many of the really awful failures I've had have been when I scrounged someone
> elses competent cells, that were not DH5 or similar.
........
> -- 
> Cheers,
> 
> Martin 
> 
> NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
> NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
> NN  NN NN           Christchurch School of Medicine            ZZZ
> NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
> 		Phone (64-3)364-0880  Fax (64-3)364-750
>

Well, I have been using HB101 for a while.  It works just fine.  Here is
the trick: Denature your plasmids as soon as possible and keep the dry tubes
at -20C for future use.  If the endonuclease really bothers you, a phenol
extraction will take care of it.  As a matter of fact, I prefer HB101 as a
host because cells are easy to resuspend into the lysozyme buffer.  This makes
a big difference when you do a large number of minipreps.  Good luck!

JC

jchen at calshp.cals.wisc.edu
Department of Food Microbiology and Toxicology
University of Wisconsin
Madison, WI 53706



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