2 replies to this topic

[Spliffeh]

Hy people,
does anyone know the concentration of salt and primers the methprimer software uses to calculate primer Tm? Because I ordered the primers I got from the software and I had put it at 60°C Tm. However on the datasheet of eurogentec it gives me Tm's of about 40°C. And if I fill it in in a primer Tm calculator it still gives me ridiculously low temperature with the concentration given on the kit!

Edited by Spliffeh, 19 February 2010 - 05:22 AM.

[HomeBrew]

I've never used methprimer, but there is considerable discussion on the Tm values in the paper that describes this software.  The software is based on Primer3, which uses 50 millimolar as the default salt concentration.  Could your difficulty be due to the software's internal conversion of non-CpG 'C's in the sequence to 'T'?

[Spliffeh]

Yes sorry I forgot to mention that, I've used it to develop BSP primers so there are no CpG's in the primerstrand. But the salt concentration might be the problem... 50mM is ridiculously high. Which kit uses 50mM anyway??? I have just checked 2 realtime kits (1 from Roche and 1 from Biorad) and 1 normal from promega and the MgCl concentration is always between 1.5 and 4mM!!! I have recalculated with an external program and my primers give indeed a Tm of 60°C if salt concentration is 50mM. This is ridiculous...