re cycle sequencing

skspoidn at skspoidn at
Thu Jun 3 05:03:47 EST 1993

re problems with Strep sequencing...

I have only ever used the Promega Fmol kit, never the USB for cycle sequencing,
 but I am very suprised that the USB kit conatains a 'labelling' cycle prior to
 the termination cycle. With the Promega kit, 1 set of cycling reactions does 
all. Given the effieciency of Taq up around 60-70 degrees, what is to stop the
 so caled labelling reactions from extending way up to the 100's bases range,
 or the Taq losing potency (do you add more for the second set of cycles?)

Anyway, the whole protocol looks wierd to me.

As far as the GC content goes, it shouldnt make to much difference,
 but the first thing I would try is increasing the 95 incubation time.

Mike Poidinger
Dept of Microbiology
University of Reading

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