sequence peters out after 150 bp

Arash Arash
Sun Sep 29 06:17:45 EST 1996


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
> ____________________________________________________________________
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> ____________________________________________________________________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




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