PCR program - strange extension step (Aug/17/2009 )
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?
3 min 94°C
30 s 94°C
60s 72°C (loop back to step 2 30 times)
30 min 60°C
Thanks for any ideas...
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.
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).