Big Dye-based sequencing - why these parameters?

Victor L. levenson at northwestern.edu
Wed Nov 7 12:59:41 EST 2001


Have you tried ABI tech people? In my - unrelated - case they were
very helpful.

Cheers,

Victor


On Wed, 07 Nov 2001 01:17:51 GMT, dk at no.email.thankstospam.net (D.K.)
wrote:

>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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