High temp sequencing

GRGGTA at PATERSON.CHRISTIE-HOSPITAL.MANCHESTER.AC.UK GRGGTA at PATERSON.CHRISTIE-HOSPITAL.MANCHESTER.AC.UK
Wed Aug 4 04:04:00 EST 1993


Jon Jafari writes

Sorry if this is a FAQ, but I couldn't find a FAQ for this group.  

I am having serious proplems with secondary structure and would like
to know if anyone has used taq to label at a higher temp (or
sequenase) with success and if so what were the conditions?  I have
called Promega re their taq kit but they "dogged me like toto" and
sent the same information that was in their catalog.  Thanks for your
help.

                                888888888888

I too have had similar problems and also plan to resort to high
temperature polymerase reactions as a potential improvement. However, I
have not yet had to as I have found that sequencing the opposite strand
of a bit of recalcitrant sequence helps as does warming the reaction to
 37 degrees C
after annealing, (ie USB kit, double stranded sequencing)and then letting
it cool to room temp prior to adding sequenase.
This is OK for the relatively small amount of sequencing I do, but carrying
out the reaction at high temp should save all the hassle.
Graham Atherton 



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