We have been operating a DNA sequencing core facility with an ABI 373 for
a couple of months now and are always using the Taq DyeDeoxy protocols.
We generally have nice reads, but sometimes stumble on problematic
templates. For instance, mostly on PCR fragments, we will get very nice
reads with one primer, but no readable sequence with the other primer,
using the same template (both primers are the same that were used to
obtain the PCR fragment). Our hypothesis is that secondary structures
hinder the sequencing reaction on the problematic strand. Realizing that
since the initial PCR worked in the first place, the problem might be with
the lower extension temperature recommended in the ABI protocol (60C for
DyeDeoxy sequencing instead of usual 72C for regular Taq PCR).
Has anybody tried to sequence with ABI DyeDeoxy kits at higher extension
Any suggestions for improving our results in such cases?
Thanks for sharing your experience, we will summarize the answers received