Vector problem (pCR 2.1 TOPO)

Per-Johan Meijer meijer at crc.dk
Fri Feb 6 19:22:38 EST 1998


Hi!

I have encountered a problem when I have tried sequencing cDNAs from
a PCR reaction ligated into the pCR 2.1 TOPO vector (Invitrogen). No
matter in what
direction we sequence the signal is very strong and nice in the first
part of the sequence, which corresponds to the vector. However, as soon
as the sequencing reactions reach in to the cDNA the signal drops, some
times to a level from which it is impossible to extract any data.
Sequencing the
same cDNA library which has been cloned into an pUC based vector
never gives these problems. The cDNAs does not have a high GC-content.
Has anybody experienced a similar problem? We use a ABI 377 sequencing
machine.

Thanks,
PJ Meijer






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