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Mini-Prep Blues

Martin Kennedy mkennedy at chmeds.ac.nz
Tue Jul 12 22:21:18 EST 1994


In article <genecutl-080794200752 at kos2mac23.berkeley.edu>, genecutl at mendel.berkeley.edu (gc) writes:
> 
> 
> I've been using a boiling-lysis protocol for mini-preps and it used
> to work beautifully.  The protocol involves vortexing the cells in
> STET buffer, boiling, spinning down the precipitated crap, then
> EtOH ppt the sup.
.......
> The problem is now the mini-prep gods no longer smile on me and I'm
> lucky to get a single sample to work out of twenty.  What can have gone
> wrong?  Please don't tell me I have to go back to phenol/chloroform
> hell.
> 

Have you changed host strain, or scrounged someone elses competent cells?  
Boiling lysis works beautifully, and plasmids sequence well, provided you use 
an endonuclease-1 mutant like DH5 alpha. Any other strains give you DNA that 
can degrade, and is not sequencable.  This isn't folklore - take it from me, 
it's FACT!!!
 
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)364-0750



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