ABI PRISM BigDye SEQ problem!!!

Mart Speek mspeek at tamm.eenet.ee
Sat Feb 6 04:48:00 EST 1999


Hi SEQUENCERS!

I am having a problem when using T3 & T7 primers (20-22 mers with
Tm ~55-65 degrees centigrade) with ABI Prism BigDye Terminator Cycle
Sequencing Ready Reaction Kit for sequencing plasmid DNA (KS) by running
standard PCR with annealing temperature 50 degrees (recommended by P-E)
and ABI 310 machine. Don't get any sequence!

Has anyone seen similar problem before?

In fact, I did get a good sequence with these primers when using
a dRhodamine Kit from the same manufacturer.

I cannot see any good reason why BigDye should not-to-allow
to get sequence with AmpliTaq DNA polymerase FS and T3 & T7.

Meanwhile I am going to reduce the annealing temperature in
PCR reaction to see whether this has some effect. I'll post
the results if there is some follow up.

Mart Speek (ESTONIA)




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