Primer Annealing Temperature

[magicsiew]

My set of primers with the forward primer with 53.0 degree of Tm and reverse primer with 64.8 degree of Tm. It is said that normally annealing temperature should be 5 to 10 degree below the primers Tm. However, in this case, even after I minus 10 degree of temperature from the reverse primer, it is still higher than the Tm of forward primer. Any suggestion on this problem? The primers were found from a literature, but inside there dint state the annealing temperature.

[JackBean]

I think it could be about 3°C lower then Tm of primer with lower Tm

However, I would guess, you have some flanking regions, right?

[magicsiew]

Means I can try with 50°C? How do check if I have some flanking region or not?

[JackBean]

yeah, I would try between 50-53°C

just anneal the primers to your sequence and you will see, whether the are 100% homologous or not. I had the same case just recently.

[magicsiew]

I tried with 55°C and it works.

[hashemyemen]

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

http://en.wikipedia.org/wiki/Polymerase_chain_reaction

[JackBean]

yeah, I think that's what we said