PCR sequencing problems
mrieder at u.washington.edu
Fri Oct 24 05:05:11 EST 1997
Yes, we have had the exact same problems in our lab. We have seen a
continual decline in our sequencing quality the past month or more and
finally have stopped using the shrimp/exo I treatment completely. After
going back to standard gel purification our results are great again. I
assume you are using the Amersham PCR reagent kit (Cat # US70995). I
suggest we call them and register our complaints. Glad to hear someone
else is experiencing this and its not a lab problem.
Mark J. Rieder, Ph.D.
Dept. of Molecular Biotechnology
University of Washington
Seattle, WA 98195-7730
On 20 Oct 1997, sequencing wrote:
> I have been sequencing short to medium lenght PCR products successfully
> (on a 377 ABI) up until a few weeks ago. I use shrimp alkaline
> phosphatase and exonuclease I as a cleanup.
> Recently all PCR sequencing has come to a halt. I get only short blips
> on the gel image. As well, the controls have much higher background,
> and more N's than normal.
> I have tried increasing the DNA and ethanol precipitation of the DNA to
> remove the inactive enzymes, to no avail.
> Has anyone else had this problem?
> Are there any suggestions?
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