Hi,
I got a PCR program that has as final extension step (at least I interpret it as extension step) 30 minutes at 60°C. Normally I use 10 minutes at 72°C...
So what is the aim of this long time at 60 degrees?
(Complete program:
3 min 94°C
30 s 94°C
90s 58°C
60s 72°C (loop back to step 2 30 times)
30 min 60°C
Thanks for any ideas...
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2 replies to this topic
#1
hobglobin
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Posted 17 August 2009 - 03:18 AM
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#2
noelmathur
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Posted 17 August 2009 - 06:07 AM
Frankly, quiet odd to my knowledge. I always do as you say.
But why do you need annealing time to be for 90sec?, 30 sec is enough, right?
Would love to hear what others have to say.
But why do you need annealing time to be for 90sec?, 30 sec is enough, right?
Would love to hear what others have to say.
#3
swanny
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Posted 17 August 2009 - 05:16 PM
AT 60 degrees, most of the thermophilic polymerases would only work very slowly. Perhaps it's there for some kind of proofreading (wouldn't work with Taq, though), or to ensure all amplicons are full length (but your point of 10 minutes @72 deg would do the same thing).
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