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Mini-Prep Blues

Dennis Templeton djt2 at po.cwru.edu
Fri Jul 15 16:52:02 EST 1994


In article <1994Jul13.152118.554 at chmeds.ac.nz>, mkennedy at chmeds.ac.nz
(Martin Kennedy) wrote:

> 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.
 
> 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!!!
>  

All that is true, but since DH5a suck in terms of growth rate and stability
on a plate, we still like HB101. The MP we use (and have posted here
several times) is a boiling prep that adds a CTAB precipitation to remove
the endonuclease and almost all of the RNAse products. This MP is *much*
cleaner than our neighbors who uses the normal boiling prep with DH5a, and
it still uses only one tube.
-- 
Dennis J. Templeton
CWRU School of Medicine



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