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Cycle Sequencing conditions

Joseph Cook cookj at bms.com
Tue Mar 16 18:53:03 EST 1999


I believe the longer extension times are due to the fact that the BigDye
mix contains Inosine in place dGTP.  Inosine is not efficiently
incorporated by the enzyme so you need longer extension times.  ABI has
a dGTP BigDye kit which requires shorter extension times.  If you use
this kit, however, you run the risk of having compressions.  If your
templates are not particularly GC rich, you may get good results with
this kit and with much shorter cycling times.

Joe Cook
Bristol-Myers Squibb  

Barbara Si mionati wrote:
> 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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