Sequencing Woes

Martin Hirst hirst at interomex.com
Fri Sep 13 14:39:30 EST 2002

On 9/12/02 7:29, "Julie McCollum" <julie at sorggenome.tamu.edu> wrote:

> 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
> College Station, TX

You may want to check if primer can anneal to more than one position on your
target plasmid (doesn't have to be an exact match either!).  Increasing the
annealing temp. on your cycles may help.





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