But I usually follow the rule: Tm-5 for the annealing temperature and it worked fine so far. I will try to do at the annealing temperature of 69 and above! By excluding, do you meant the annealing step right for the 2 step program?

Hi Shahid,

Try to use the Tm Calculator from NEB. And Only use the part of the primer that anneal to your template to estimate the Tm of your primer pair. Furthermore, follow the suggestion for genomic DNA or chromosomal DNA when using Phusion polymerase HF. ~40 s/ kB. And don't use to much template DNA. Sometimes a final volume less then 30 µL gives error or no products. To clear it out: I think your linker is not part of your template SO DON'T INCLUDE THIS PART WHEN ESTIMATING THE TM.

try this program

98 30s

30x cycles

9810s

Tm 30s

72 30-40s/kB (even if Tm is 72, do them separately!)

72 10min

4 hold

Good luck,

TS

That's right in principle, but: The product of the first cycles becomes the template for the later cycles -- and that product includes the linker. As the cycles progress, the original template becomes crowded out rather quickly by linker-containing product. So in the later cycles, your entire primer will anneal, including the linker.

I've had great success with stepping up the annealing temperature to reflect that. For the first 2-5 cycles I anneal with Tm calculated without the linker, and then the following 30 cycles with Tm calculated with linker. Makes a big difference in my hands. I have a lot of reactions that won't work in any other way.

Thank you very much for such valuable discussion and I am definitely going to try this out on Monday :)