Tm of the primers - company sends primers with lower Tm than expected (Oct/31/2008 )
I usually design my primers using primer-BLAST or Oligocalc.
In a previous primer order, although the theoretically calculated Tm of the primers using the above software was 60 C, the certificate of analysis of the primers that I received had a Tm of 50 C.
It seems that the companies use a different algorithm to calculate the Tm than I do.
Which Tm should I trust? I mean, the Tm in the certificate of analysis is practically tested or they just calculated the Tm like I did, only with a different primer tool?
I am about to conduct an experiment in which high Tm of the primers is needed (<70 C), so I need to be sure....
Thanks in advance!!!!
I am using MeltCalc a Excel Macro.
Is everytime very close to the companies one.
There's no way the primer synthesis company empirically derives the Tm of your primer -- they are just using a different equation than you. There are three major equations, and several slight variations thereof -- the "Wallace rule" equation, the "salt adjusted" equation, and the "nearest neighbor" equation. The latter is considered the most accurate, but all are just theoretical and only valid for certain oligo lengths.