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Help with primer design


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3 replies to this topic

#1 borriello.87

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Posted 24 March 2012 - 05:35 PM

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!!

#2 pcrman

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Posted 24 March 2012 - 06:25 PM

>>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.

#3 borriello.87

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Posted 25 March 2012 - 02:51 AM

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!!

#4 Trof

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Posted 26 March 2012 - 02:39 PM

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.

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