reomi, on Apr 21 2009, 11:17 AM, said:
The forward primer includes a PstI site, tm 61, 39% GC (aaaCTGCAGATGATATCTGTCACAGATATTCGTAGAGCGT). For the reverse primer, The inclusion of a myc tag (lower case) and a NotI (in italics) site, while trying to optimize the tm and gc content of the annealing region makes for quite a long primer (80 bp, ATAGTTTAGCGGCCGCcttaaaggtcttcttctgagataagtttctgttcTCTGATTATTGTCTAATATGGTCTCTACGC
). The annealing portion of the primer is in bold, 30bp long, 37%gc, tm 58. Any more or less sequence specific and the GC% drops quickly.
The sequence prior to the restriction sites is what the NEB guide suggests. Using an annealing of 55 to 59 degrees with Phusion, I get no amplification.
As I understand, the annealing temperature or Tm should base on those bold sequences, because that will be the region actually anneal to your template during PCR. Thus, you should consider the Tm of your forward primer as 57.5 and the Tm of your reverse primer as 55.9 (calculation base on the computer tool I use).
As a result, annealing temperature of 55 to 59 could be too high for those primers, and maybe you should consider reducing it to 50 to 56.
Since the size of your reverse primer, the PCR efficiency with this primer will be low no matter what temperature you use.
I assume you would like to use this PCR product for downstream application like cloning. I have similar task before, and to get a usable product, I was given the following suggestion:
First, try 10 to 15 cycles of PCR only use reverse primer at low temperature.
After first PCR complete, add additional PCR mix with both forward and reverse primers (yes, higher reverse primer concentration). Run PCR as you usually run with same lower annealing temperature at first 5 to 10 cycles and then increase annealing temperature to appropriate one (in your case probable between 50 and 55 or higher) for the rest of PCR reaction.
In my case, I did get significant improvement; hope this will help your case.