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Mini-Prep Blues

Michael Benedik bchs1b at Jane.UH.EDU
Sat Jul 9 21:47:53 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 pellet I got from this has a huge amount of protein in it, but,
>surprisingly, my restriction digests always looked great and I even
>had much greater success sequencing this DNA than DNA from alkaline
>lysis preps.
>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.
>
>
>
>
>-- 
>--gc
>
>I know you like rock 'n' roll. Have you considered overdosing
>on heroin, like many of your pop-star favorites?  --Life in Hell

Have you changed host strains by any chance? The boiling minipreps
leave endoA intact which can chew up your DNA. So you need to either
1) use an endoA mutant strain or
2) if you use and endoA+ strain then do NOT treat with RNAse
until after the restriction digest. RNA inhibits the endoA 
activity.

Other possibilities are: lysozyme gone bad, temperature no
longer what you think it is, new batch of STET buffer contaminated 
with something, etc etc. But we still use boiling prep all the time
and love it.

----------------------------------------------------------------------
 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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