Mini-Prep Blues

Tracy Aquilla aquilla at salus.med.uvm.edu
Fri Jul 15 13:21:50 EST 1994


In Article <genecutl-080794200752 at kos2mac23.berkeley.edu>,
genecutl at mendel.berkeley.edu (gc) wrote:
>
>
>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

    Well, here's my $0.02 worth. I happen to like the boiling method, and
have used it as my standby for a long time. If the mini-prep lysis buffer
gets old, the triton-x100 goes, and you need to make it fresh (every month
or two is best). Secondly, do you get good lysis? If not, the lysozyme may
be old also. While some *strains* do not lyse well by boiling, if you
haven't changed strains, this is not your problem. My guess is that you need
to make new lysis buffer. This technique occasionally gives me the same
problem you are having, and making new buffer always fixes the problem.
                                    Tracy
Tracy Aquilla, Post-doctoral Research Associate
Department of Molecular Physiology and Biophysics
University of Vermont, College of Medicine
aquilla at salus.med.uvm.edu



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