difficult PCR - (Mar/13/2006 )

I'm about to attempt PCR deletion mutagesis to delete ~150 bp from a ~900 bp restriction fragment. The relative (predicted) stability of primer binding to the two regions to be fused requires me to use unusually long primers (up to 41bp).

I'm wondering if anyone has tried this, and what problems I can anticipate, hopefully even how I can do a little preemptive troubleshooting.

Cheers,

Megan

---

No answers yet, so i give my analysis even if it's not very helpfull.

Start by trying to calculate annealing temperatures (or let your primer program do the work).
It's a long time since I last did such things, but I think the formula was 2xA +2xT+4xC+4xG or something in the neightbourhood, and the lowest possible result for a 41 b primer is 82C, which is much higher than a the action temp for Taq.

I would say; will be difficult task, and that's before we start thinking about everything else.
Please correct me if I am completely wrong.

---