ABI sequencing artifact

Lisa Bibbs lisa at scripps.edu
Mon Sep 19 12:04:21 EST 1994


> > Message-ID: <quinnt-1409941902420001 at microb3.biostat.washington.edu>
> > NNTP-Posting-Host: microb3.biostat.washington.edu
> > 
> > I have been using the ABI DyeDeoxy kit with Taq for sequencing and have
> > encountered a strange artifact.  On one particular template, I get
> > beautiful read for about 200 bp, then spotty read for about 10 bp, then
> > absolutely nothing for the rest of the run.  This has happened twice, and
> > the break always occurs in the same spot.  I was wondering if anyone had
> > any similar occurences or any ideas about what's going on.  Thanks a
> > lot!!!
> > 
> > Matthew Stoecker
> > U. of Washington


How does your control look?  

We have seen this several times.  It is never in our control.  Upon
checking, it seems as if we can sequence up to the cloning site.  (Has
happened several times with PET vectors.)  In those instances we have been
able to come in from the other direction and sequence to the same point. 
My guess has been that there is some type of secondary structure that we
can't get through.   

We have also seen it with samples that have hit a GC stretch.  In those
cases we add Triton or DMSO(1 uL) and raise the denaturation temp 2 C.  The
read isn't great but we can get an oligo to continue.

Good Luck.
Lisa Bibbs



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