Sequencing Woes

Julie McCollum julie at sorggenome.tamu.edu
Thu Sep 12 09:29:21 EST 2002


We have recently (within the last month or so) started having problems
with our sequencing.  The sequences have double peaks which stretch
along the entire length of the read, including the vector sequence
(usually pBS SK-).  Basically, they look like double picks, except
that, at least in my hands, double picks usually have clean sequence to
the cloning site, and then start to have double peaks.  If this were
only happening in a few sequences here and there, I could live with it,
but it's whole plates.

Pertinent info:
1.  Plasmids are being prepped using Promega's Magnesil system
(magnetic bead system for lysate clearing and plasmid purification) on
a Biomek2000.  This system has worked well previously.  We have changed
out all solutions in case of contamination and have the same results.
2.  We are using Big Dye v. 3.0 and running the reactions on an ABI
3700.  The capillaries and all reagents are new.
3.  pGem control sequences look great.  These were done at the same
time as my other stuff, with the same master mix, in the same plate.
This seems to rule out both the 3700 and the cycle sequencing
reactions.
4.  My boss is doing several preps today, manually, of some of the same
plasmids to rule out or point to the plasmid prep.

Has anybody seen these types of results?  If so, what caused it and
what did you do to fix it?

Thanks for your help!

Julie

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




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