PCR sequencing problems

M. Rieder 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
Box 357730
Seattle, WA  98195-7730

Phone: 206.685.7339
Fax:  206.685.7301

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?
> 
> Jan
> 
> 
> 





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