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
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.