2 replies to this topic

[E Henry]

Hey gus,

I am new to qPCR. I used Primerquest from IDTDNA to design the primers for qPCR. The Tm parameters were set as 60-64C, 62C optimal. However when I check the primers IDT recommended, Tm is around 55C (by manually adding up G/C=3 A/T=2). A regular PCR was performed with annealing T as 60, only 3 of 10 got bands (right ones at 100-150 bp and unspecific ones at 400 bp and top).

Double checked with IDT website, it claimed that the latest concept of calculating Tm is to integrate consideration of salts in reaction buffer thus when using their software to design q-pcr primers Tm will be calculated based on standard qPCR parameters (50 mM K, 3 mM Mg, 0.8 mM dNTPs). Does this mean a primer with Tm of 55C in 50 mM NaCl will work as Tm of 60C in a q-PCR? I am going to use Power SYBR® Green PCR Master Mix from AB which the salts concentration is not released. Getting quit confusd now.

Does anybody get this problem before or clear out the mystery. Any thoughts are appreciated.

Henry

[ElHo]

I usually try different annealing temperatures around the calculated Tm to check which one works best with my pcr mix (biggest amount of pcr-product without unspecific binding). You can never tell whether a calculated Tm will work for your specific pcr or not. If possible use a sample with confirmed expression for your gene of interest (assuming you´re doing expression analyses).

[E Henry]

ElHo, on 10 August 2010 - 12:35 AM, said:

I usually try different annealing temperatures around the calculated Tm to check which one works best with my pcr mix (biggest amount of pcr-product without unspecific binding). You can never tell whether a calculated Tm will work for your specific pcr or not. If possible use a sample with confirmed expression for your gene of interest (assuming you´re doing expression analyses).

Thanks ElHo, I am going to try temperature gradient like you suggested. The follow up question is that do I have to choose the primer set with optimal annealing T at 60C in order to use in ABI 7500? I am going to check expression of 8 genes out of 27 tissue samples. A standardized annealing T is required which is also suggested in ABI manual.