Unusual manual sequencing problem

W. Travis Johnson johnson1 at primenet.com
Wed Oct 2 23:10:11 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.

>Thanks for any reply!

>Caroline GUIHAL
>Scientific and technical services Department
>Royal Botanic Garden Edinburgh

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

Is it possible that your terminators have been cross contaiminated
(bad stocks or inadvertant of same pitette tips for different
dideoxynucleotides) ?.  Therefore, the fact that the bands disappear
later could be due to an extremely low concentration of the
contaminants that diffuse with time.

Johnson




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