sequence peters out after 150 bp

Koen. A.L. De Smet k.desmet at ic.ac.uk
Fri Sep 20 08:23:57 EST 1996


In the last few weeks, we have had lots of sequences that started off with
very nice high peaks, but peter out after 100-200 bp.

We always use the ABI310 Gene Scan DNA sequencer (yes that is the new
capillary one that is hardly ever mentioned in this newsgroup) and the ABI
dye terminator Ready Mix with the AmpliTaq FS enzyme. This would normally
give us around 300 bp of sequence.
Most templates are plasmids prepared using the Hybaid Recovery spin
columns, but we have had the same problem with some PCR products as well.
In most cases, we used the universal primer.
Using the pGEM template in the kit, we get good results, which suggested
that it was a template problem. But when sequencing some old templates that
worked a few months ago and were stored in the freezer, we still have this
problem. Cleaning up plasmid preps through a microcon filtration unit
didn't improve things either.
Changing the capillary and buffers or cleaning the machine didn't help either

We are now a bit stuck, in that we don't know which parameters to change.
Does anybody out there have any suggestions on how to solve this problem?

Thanks already for any answers!



Koen



____________________________________________________________________

        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain

        Tel: (+44)-(0)171-594 3946
        Fax: (+44)-(0)171-262-6299
        Email: k.desmet at sm.ic.ac.uk
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