Question about PCR reaction condition - (Jun/14/2006 )
I am trying to do PCR with a cDNA. The desired sequence is 1.7 kb. Due to some limitations I have to use a pair of primers whose Tm are around 72 C. I heard that the annealing temperature during the PCR cycle should be about 5 C lower than the Tm of the primers. That would make the annealing temperature about 67 C. Usually the PCR cycle is "Denature 94 C 30'', Anneal 55 C 30'', Extend 72 C 1'". If the annealing temperature is 67 C, what temperature should I use for extension?
Any suggestion or advice would be really appreciated!
72C - normally.
BUT Check with the enzyme manual. I have polymerase which extends at 68C.
extension temperature depends on the enzyme youare using. 72 C is the most widely used extension temp. for Taq DNA pol. you can try the same extension temp.
I have repeatedly and succesfuly used 2 step PCR, where you cycle between annealing and denaturation, without extension step.
All the amplicons I wanted were below 300 bp so I don't know for longer fragments, but it saves a lot of time and worked fine (I used a final extension step at 72ºC)