Two design programs give very different primer Tm values - RT-PCR (Jun/26/2006 )
My 134 ProbeFinder™-designed (Roche/Exiqon) primers varied in melting temperature from 53.2 - 63.2C, according to the company I ordered primers from. Melting temperaturs mismatched to a maximum of 8.3 degrees Celsius for primer pairs.
What is the most consistent Freeware program for re-designing primers so they are very close to a 60C melting temperature, once ProbeFinder™ optimises their location on a transcript?
Thank you very much!
The difference is due to the formula they use to calculate the Tm. I found the MWG calculated Tm very good. You can register and do as you were ordering primers: you put the sequence of your primers and press check; it will tell you which is the melt temp... and then you don't order from them if you don't want. I think it was reliable and I never had problems with PCR or realtime
I usually try to find the Tm calculated by the company where I buy the polymerase (for example, I use Finnzyme's Tm calculating tool for phusion amplification reactions)
it usually works well for me
Thank you VERY much for your feedback. I buy primers from MWG, so I will do this.
I have contacted Roche, attaching the MWG primer melting temperatures, and they are very interested in this discrepancy. I may have triggered them to upgrade their primer program, (at least this is what I am hoping).
It is no secret that ProbeFinder™ uses primer3 for designing primers. It says so on the website if you look hard enough
A version of primer3 where you can play with a lot of parameters is available at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
I trust primer3 because the program is easy to work with and the given Tm`s are reliable.
You can f.ex. chose the max. Tm difference of your primer pair or the max. self-complementary of the primers which will help to avoid the formation of dimers.