Traditonally, PCR annealing temp. is run at approx 5 celcius below the melting Tm of the primers used. Is there a reliable program to use to determine the melting Tm? I used two programs found online, and got two different Tm's for the following primer: ctc aca act tag tcc tcg gtt gat ggc
web-site 1 Gives me 61.3 C
web site 2 Gives me 77.8 C
I did a gradient temp PCR from 45 - 58 C, and the result was non-specific amplification (more than one band). I am going to run another gradient temp PCR starting at 58 - 70 C. I suspect that the first web-site is not giving me an accurate Tm calculation for the primers. I would hate to do the calculation on my own every time I design a primer!
0.025 U/uL Taq
0.25 uM primers
2 mM MgCl2
2.5 mM dNTP's
approx 1 ug template genomic DNA
I would post the URL's for the sites, but I guess I am not allowed to do that yet since I am a new member on this site
P.S. could it be that there is a lot of template?
Any feedback will help. Thanks