Calculating Tm for bisulfite primers? - (Jun/13/2006 )
I am trying to design a PCR for the primers I chose. I first tried primers that I got from MethPrimer and based my Ta on the Tm given by methprimer. However when I plug these same primers into online Tm calculators like the one provided by promega ()
I get WAY lower Tms than those provided by methprimer.
Why the differences and what should I trust when deciding on the initial Ta?
Thanks for any advice!
the differences are due to how the Tm was calculated, there are a number of formulas to do so, also the assumed salt concentrations for the calculation would have an effect on the results. as you used two different calculators, they will almost for sure give you different results. I would chose one and stick to it.
Primer express is pretty accurate. (try methyl primer express! it's free)
Thanks. I knew the salt concetrations would change the Tm but I didn't realize that it could be so drastic. I'm seeing differences of 10C sometimes.
Looks like I'll just pick one a try it out,
the difference would then boil down to the formulas used to calculate Tm, there are many out there.