PCR primers with high Tm - (Jun/01/2006 )

Hi,
Has anyone successfully performed PCR with primers with very high melting temperatures (above 75C - I have one as high as 83C)? If so, does using a very high annealing temperature affect what extension temperature you use, or will 72C still be OK?
Any comments would be really helpful.
Thanks

Hi

You could try 2-step PCR, I mean that you could combine the Annealing and Extension Steps into one at72C!

Good Luck!

is it a bioinformatic calculated tm or the nearest neighbour method? (4 GC 2 AT)

one of my lab mate done successful pcr with primer of Tm 74 deg

I've done good PCR at 77, but never higher

just used two-step method with Phusion

98 60s (one cycle)

98 15s
72 90s (25 cycles)

72 5 min (one cycle)

worked great

I did PCR on 85% GC content DNA, and it worked OK. I used really short primers (15mers had a TM about 75C), plus DMSO or betaine. The reaction worked really well as a "normal" PCR reaction (whatever normal is!!!).
In order to reduce mispriming events, I did a touchdown PCR.

Good luck