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salts in PCR


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4 replies to this topic

#1 criscastells

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Posted 10 October 2011 - 07:30 AM

Hi everybody,

I am looking for some different primers and I want to analyze them between choose. I have analyze dimers and hairpins but I have a little problem with the Tm. I am using "oligoanalyzer" and it only takes into account the Mg concentration and Na concentration. It may be OK, but the buffer of my polymerase contains NH4, K and Tris and I don't know if they may affect to my Tm.

Does anybody knows what happen with this? Its really important to analyze the primers?

Thanks you very much

CRIS

#2 hobglobin

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Posted 10 October 2011 - 08:36 AM

Cations stabilize the duplex state of DNA, and the algorithms for salt correction formulas are developed using Na+concentrations, for whatever reasons. So I'd take into account monovalent and bivalent ions as primer3 does it. I guess it's not that important if it's K+ or Na+ (someone might correct me here).
NH4+ surely also will have an effect as it is a cation, but it also influences the enzyme's activity. I don't know any program that accounts for that, but perhaps no data are available, as such additives are anyway a trial and error issue.
Actually my way to deal with it is to calculate roughly an annealing temperature (using standard settings) and then later doing a gradient PCR, if the reaction works. The calculated Tms are also always an estimate that have to be verified and then optimised. And also with simple formulas such as
Tm (°C) = 81.5 + 0.41(%GC) - (675/N) or Tm = 4(G + C) + 2(A + T) °C PCRs worked...Posted Image

Edited by hobglobin, 10 October 2011 - 08:39 AM.

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#3 criscastells

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Posted 11 October 2011 - 01:53 AM

Thanks you very much for your advice. It makes sense :P

#4 phage434

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Posted 11 October 2011 - 10:21 AM

The main thing to know is that (1) the calculations are inaccurate and (2) it doesn't much matter. PCR reactions are robust and if primers are vaguely reasonable will work over a wide range of annealing temperatures. Use primers of 18-24 bp (higher if low GC, lower if high GC) where the length is the portion of the primer binding to the template. Try the PCR reaction with annealing at 55C. If it doesn't work, try gradient. If that doesn't work, try designing the primers again, or analyze them with the tools at idtdna.com for hairpins, homodimers, and heterodimers. Strong binding in any of these leading to a recessed 3' end bound to a complementary region will give trouble.

#5 criscastells

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Posted 17 October 2011 - 05:39 AM

Thanks again for your help I will work on it




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