Unusual manual sequencing problem

c.guihal at rbge.org.uk c.guihal at rbge.org.uk
Wed Oct 2 05:48:28 EST 1996


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.

Thanks for any reply!

Caroline GUIHAL
Scientific and technical services Department
Royal Botanic Garden Edinburgh

e.mail: C.GUIHAL at rbge.org.uk




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