PCR reactions - What condition should I use? (Oct/11/2005 )
I intend to run a PCR (a site directed mutagenesis) and I found several protocols but none of them had the same conditions!
I would like to know the importance of : - the number of cycles (going from 10 to 33 cycles)
- the third temperature ( between 68 to 72 °C)
- the time of the third temperature ( 45sec to 2min)
thanx for your answers!!
i can add some infos, but probably the protocol will differs.
first, what do you call third temp? I assume this is an elongation temperature, and i've always saw (regarding polymerases i use) 72°C
for denaturing, it's 95 or 94° (94 for phusion pol) and times varies. 1' in general (45'' for Pfu Turbo pol)
Annealing temp is tm - 5° (Tm : 4 for numbers of G/C + 2 for A/T) or this web pages for oligos longer than 20.
Elongation is 72°, and generally 1' per kb. Except for Pfu, 1'30'' per kb.
Here is my protocol, that goes well.
hope that helps to clarify your situation?
Pfu has strand displacement @ 72, but not @ 68, so you ebtter use 68? (Stratagene advises this). Also, for long templates, you mostly have to use 68 anyway.
Try to keep the number of cycles low, I generally do 18, which is plenty.
For more information, go to www.stratagene.com and check out their manual for quickchange site directed mutagenesis.
thanx for the replies!
I understand now why 72 or 68°C for the elongation (polymerase)
but sometines I just see 90sec then i find 2min/kb of plasmid (mine is 5.4kb) another one just 45sec!
oh and one of my primer is 100nt!!!
Although we generally say that 1kb/min for polymerase, but in fact I found sometimes less than 1 min was able to give me product at 3.5 kb (the polymerase finish one whole circle around the plasmid!). However, I still suggest you follow 1kb/1min to calculate the elongation time for your own reaction, as it will be more guaranteed.
You also mentioned your primer is 100nt. That was too long for mutagenesis, all your primers will bind with each other before they can anneal to the plasmid.
thanx for your replies I'll try and hope it'll work!!