Unusual manual sequencing problem

Rico Laage rico.laage at urz.uni-heidelberg.de
Fri Oct 4 07:23:47 EST 1996


c.guihal at rbge.org.uk wrote:
> 
> We have come across a strange phenomenon: On our latest
> autorads some bands appear across all four lanes on the
> acrylamide gel.
> For these reactions, a PCR product (which had been sequenced
> succesfully before), was sequenced with eight primers (four
> on both strands) using the Sequenase PCR product direct
> sequencing kit (Amersham).
> Other DNAs have been already sequenced in this domain with
> the same primers, and did not show this phenomenon. After
> comparing the old and the new autorads it is clear that it is
> the G bands that appear in four lanes!
> However, after about five hours of migration, these G bands no
> longer appear in all four lanes and the final part of the sequence
> can be read as normal.
> Reactions have been made following the protocol described in
> the Sequenase booklet, with a new stock of sequenase version
> 2.0 freshly diluted in a glycerol dilution buffer.
> I did not find any specific answer to this problem in the
> booklet, and therefore I am now looking for some help and
> advice from anyone who as had this problem before.
> 
I thought this is a typical problem for GC rich sequences sometimes you do not 
denaturate the secondary structure completely and the sequenase "falls off the 
DNA" this looks like a termination in all lanes. it helps perhaps to rise the temp.

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