unspecific polyT signal/sequence

Peter Kuhnert kuhnert at vbi.unibe.ch
Fri Feb 27 13:16:11 EST 1998


Does anybody have an explanation for my following problem?

I am sequencing a PCR product obtained by RT with polyA primers and
subsequent PCR with specific primers for my cDNA. The PCR product is
purified and sequenced with 8 primers on an AB310. With all 8 individual
reactions I get a polyT sequence which can already be seen on the raw
data: more or less flat lines exept for the red T line. The signal is
fainting away probably due to the loss of labeled ddTTP (I am using low
amount of sequencing mix). I actually know the sequence of the fragment
and it is not a polyT sequence.

I have not the faintest idea where it could result from and I have
sequenced succesfully a similarly obtained fragment before. Moreover I
already sequenced dozens of PCR fragments with no problem. Even if the
originally used polyA primers of the RT would still be in the sequencing
reaction it would not explain the result.
Any explanation is welcome.


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