PCR primers Tm higher than extension T - (May/16/2006 )

HI,

i`ve got a problem with primers. They have 77 and 82 deg Tm. What protocol can I use? Is 2 step only enough? eg. denaturation and extension ( from 95 to 72, 35 cycles?) Will it work? Anybody?


Thanks,


hady

it can work. i have seen people get results with a 2-step pcr. you would have to try to find out if it will work for you.

I prefer high tm primers to low tm primers.

Two-step amplification also limits the possibility of primer-dimerization and hairpin loops.

I think you will have success with this primer pair.

-Matt

2-step is great

hello,

so i tried to do it that way. 95 deg 30 sec and then 72deg 1.5 min.... no results. just blanks. i used for the first time eppendorf epgradient s with silvel block. it reaches 95 deg in 5 sec!!!
so the time between 95 and 72 deg was below 1 sec! maybe it didn`t have enough time to hybrydize? i will try with "normal" eppendorf...

are they cloning primers? are your high Tm's the total primer Tm's? if they are cloning primers, there are other considerations when figuring Tm...if you have introduced RE sites, or other sequence, that is not homologous to your template?

just asking to be sure, I learned that lesson the hard way once

Try an annealing/extension of 65-68C instead of 72C