Cycle Sequencing conditions

Barbara Si mionati rhteam at eos.bio.unipd.it
Mon Mar 8 18:11:49 EST 1999


Hi,
I was wondering if it is possible to modify the thermal cycling
conditions described in ABI protocols for Big Dye Termintors
The raccomended conditions are:
95°C for 30 sec
50°C for 15 sec
60°C for 4 min
In particular does anybody know if it is possible to shorten the
extension time?
If not, why, considered that in a PCR reaction the polymerase performs
about 1000-2000 bases /min? Is it because of a low extension temperature
(60°C)?
Does anyone has experimented succesfully other conditions?

   ========================
    Barbara Simionati
    CRIBI Biotechnology Centre
    and Department of Biology
    University of Padua, Italy





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