PCR quickchange with high Tm oligos - help needed (May/29/2006 )
I´ve tried several times to get a point mutated gen by PCR (following the quickchange protocol). But I´m having problems to get a particular mutation which is into a rich GC region, so the primers I have to use have a 84.8% of GC and a TM of 84.4ºC... I have been able to get another point-mutations by this method, but I am not able to do it with this primers...
Which alternative protocol could I follow with?
I´ve though a 2 step PCR or maybe add some DMSO (maybe 5% should be enough) But I´d like read more suggestions about it.
what about using shorter oligo?
5%DMSO may be ok. I've done 12%DMSO as well.
Phusion polymerase use a higher TM procedure and denatuing at 98°. May be an alternative for you.
Thanks Fred! Before ordering shorter oligos I will set up a three PCR more with 5 and 10 DMSO, and a 2 step... if none of them works, I will follow your suggestion and order shorter oligos...