pcr cycle

nsaunders at molbiol.ox.ac.uk nsaunders at molbiol.ox.ac.uk
Wed Jun 19 06:36:37 EST 1996


>Hi reader:
>
>    Recently I read some methods in the papers about the pcr cycles
>    the cycles is
>
>   95 C for 5 minutes, 60 C for 1 minute, 72 C for 1.5 minutes for one
>cycle
>    95 C for 1 minute, 60 C for 1 minute , 72 C for 1.5 minute for 26
>cycles
>     95 C for 1 minute, 60 C for i minute , 72 C for 10 minutes for one
>cycle
>  I do not understand which reason is   95 C for 5 minutes for first
>cycle  and 72 C for 10 minutes for last cycle
>Does any paper explain that ? Or somebody can explain that ? Thank a lot
>
>
>
>Shi-Jye Chu

PCR cycles can seem pretty "make it up as you go along" at times. The 1st 95 C 
step is to ensure a complete denaturation I guess; it can also be useful for 
"hot start"; you can pause the cycle at this step or a second step with 
slightly lower temp (say 85 C) then add the Taq, so as not to get extension of 
non-specifically annealed template-primers formed when the temp was rising.

The final 72 C is to fill in any incomplete extensions; it can also be useful 
for polymerases like Taq in ensuring that A-overhangs are added to the ends, 
so you can clone into a T-vector. In fact, T-vectors are made by incubating 
blunt-cut vector with dT or ddT and Taq at 72 C.

Neil Saunders
 




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