Sequencing Woes - Update

Julie McCollum julie at sorggenome.tamu.edu
Tue Sep 17 11:37:10 EST 2002


Thanks to everyone who replied!

I think we have isolated the problem to some kind of contamination
somewhere in our plasmid preps.  Last week, we did experiments which
pretty much ruled out everything but the plasmid preps.  We used our
primer (M13 Reverse) on our template and on pGEM.  The pGem looked
great, ours looked horrible.  We also tried the M13 Forward and T3
primers on  our template and pGEM and got the same results.   Looking
at the "bad" plasmids on an agarose gel, compared with the new preps my
boss did last week (he used a Qiagen prep, and a new Magnesil kit), it
seems like the new preps have lots of supercoiled DNA and a little
linear, but the bad stuff has way more linear than supercoiled, which
makes us think there is something that's chewing up our DNA in the old
kit.  We have thrown the old stuff away and are trying a prep today to
make sure last week's success was not just a fluke.

Thanks again for all your help!

Julie


--
Julie McCollum
julie at sorggenome.tamu.edu
USDA-ARS, SPARC, CGRU
College Station, TX




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