Help with primer design - (Mar/24/2012 )

Hi all,

I'm experiencing some problems using Primer-Blast to design prmer pairs. In particular, I don't know how to fill the boxes "Concentration of divalent cations" and "Concentrations of dNTPs", because I haven't found these values in datasheets of many SYBR Green PCR master mixes. I wonder whether there are commonly used fixed values for these parameters. As far as I understand, these values are important to calculate primer Tm, right?

Yet another question. Should primer Tm be slighlty higher than annealing temperature (for example, 5°C higher), or should it be equal to annealing temperature?

Thanks in advance!!

>>Concentration of divalent cations" and "Concentrations of dNTPs",
Leave them blank or use the default value. You don't need to wrroy those things in primer design

>> Should primer Tm be slighlty higher than annealing temperature (for example, 5°C higher), or should it be equal to annealing temperature?
Primer-Blast uses primer3 algorithms. the default primer Tm is usually 60C which is perfectly fine for 60C Ta.

Than you for your reply!!

My only concern is that when I pick a primer pair from RTPrimerDB (with a Ta of 60°C) and put it into Primer-Blast without filling those boxes, the Tm fall between 50 and 55°C. If I use 2.5mM as "Concentration of divalent cations" and 0.2mM as "Concentrations of dNTPs" (as far as I know, these could be realistic values in the PCR-tube), Tm rises of about 7°C. Likewise, If I design my primer pair using a Tm of 60°C, the actual Tm will be 67°C after filling those boxes.

Thus, I wonder whether the rise in the Tm could affect my qPCR and lead to unspecific amplification or it is of little importance and even a slighlty higher Tm (67-68°C) works well with a Ta of 60°C.

I hope I've been clear... thanks again!!

Try setting the Salt correction formula to 'Schildkraut and Lifson 1965' and Table of thermodynamic parameters to 'Breslauer 1986'. That will display same Tm as original primer3 algorithm (usually 4-5 °C higher that suitable Ta). I design for Tm 65 °C in this setting.