We're using PE's AmpliTaq Big Dye terminator sequencing kit
to sequence PCR products. A gel ran Friday using the same
tube of sequencing mix worked fine, but last night's was
blank except for a few dye blobs. One change was the
annealing temperature we used on our PE 9700 thermal cycler.
We had been using 48 oC but switched to 42 oC. Obviously we
did not expect to get less signal with a lower annealing
temperature.
Has anyone used 42 oC as an annealing temperature for cycle
sequencing with AmpliTaq?
Phillip San Miguel
