Koen. A.L. De Smet wrote:
>> 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> ____________________________________________________________________
> __ __
> __ Send me your opinion on our departmental World Wide Web page: __
> __ http://www.sm.ic.ac.uk/medmicro/home.htm __
> ____________________________________________________________________I
experienced a similar problem once. The solution was that I was using
too much primer. When PE's manual recommends 3.2 picomoles of primer,
they really mean it. Too much primer will result in a lot of short
sequences and will use up the tagged nucleotides in a hurry. Hope that
your problem is as simple as that.
afsh002 at mc.duke.edu
