salts in PCR
Posted 10 October 2011 - 07:30 AM
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
Posted 10 October 2011 - 08:36 AM
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...
Edited by hobglobin, 10 October 2011 - 08:39 AM.
- criscastells likes this
...except casandra's that did belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.
Posted 11 October 2011 - 10:21 AM