Can I add additional sodium ion to PCR reaction buffers? - (Oct/18/2005 )

Hi,

I'm about to do mutagenic PCR recently. My mismatched primer is 50 bases long, but its melting temperature is only 67 degrees celsius. According to the Strategene protocol, they advise the Tm should be no less than 78 degrees.

From the oligo properties calculator ( you can find it online), I know I can increase the Tm or primers to 81 degrees by increase the sodium ion (Na+) to 50mM. However, the 10X PCR reaction buffer in my lab doesn't contain Na+. It contains KCl (10mM -- final concentration), MgSO4, etc.

I'm wondering if I can add additional sodium ion to the reaction buffers. I called invitrogen for this matter, they said they didn't know, but advised me to titrate for the optimum amount of sodium ion addition.

anybody knows? or I'll have to titrate for the optimum amount of Na+??

thanks

According to QIAGEN, at 25 mM NaCl, the yield from Taq is reduced. At 50 mM, no product band is observed. (D. Loffert, S. Stump, N. Schaffrath, M. Berkenkopf and J. Kang, 1997 PCR: effects of template quality. Qiagen News 1: 8-10.)

KCl inhibits at 75 mM.

You can obtain the same effect by increasing your Mg conc. - 1mM Mg is equivalent to ~100mM Na

Daniel

Improved DNA sequencing traces

pfu doesn't like high sodium level either

Daniel

Higher quality DNA sequencing

Could you post your primers? I don't know if I believe your Tm values. What are you using to calculate Tm?

-Matt