Big Dye-based sequencing - why these parameters?

D.K. dk at no.email.thankstospam.net
Tue Nov 6 20:17:51 EST 2001


Hello, 

I have always done Big Dye cycling close to ABI recommended 
protocol - 50-55C annealing, 60 min extension for 4 min. Recently 
had a chance to think about it and realized I do not understand the 
reasons behind two things:

1. Why 60C extension? Isn't it a Taq polymerase, and wouldn't
it work just fine at ~70C?
2. Why such long extension times? Taq is pretty fast and even at
60C would certainly synthesize > 700 b/min. 

I suspect both reasons have something to do with dye terminators
being poor substrates, but I'd rather hear from someone who really 
knows. Unfortunately, nobody I talked around here could provide
an answer. 

Thanks!






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