Primer melting Tm - help

[Lina]

Hi,

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!

PCR conditions:
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

[eul]

Hi Lina,
these are some websites suggested to me from the administrator when I had the same question. All of them gave me almost the same Tm for my primers.

http://www.promega.com/biomath/calc11.htm

http://www.basic.northwestern.edu/bi...oligocalc.html

http://insilico.ehu.es/tm.php

[Lina]

Thank you

[polymerase1]

hi there,
to bare in mind Lina, there is no positive program to give you an exact Tm. because each program is using different algorithm and, therefore, you cannot have an exact adjusted Tm. Even, for instance, if you use the (A+T)2 +(G+C)4 equation, you may end up with nothing. taken together, these are different criteria to assess a certain condition and, hence, you may find it useful and sometimes are not. As you've mentioned, that's true annealing should go with 5C below the optimum Tm but that's not the usual case because sometimes you've to go 3C above the Tm to get the optimal annealing temp.
These are things to keep in mind while running your protocol.

Your both gradient protocols seem pretty fine but I would suggest 55 for 15 cycles and then throw the stone upto 68C for 20 cycles. what's your cycling conditions?????? initial denaturation, denaturation, annealing, elongation???

Yes your template is really high but try not to change many variables at a time, so i would stick with the cycling conditions and if it is still unsuccessful then you may start changing your recipes.

don't forget to put your cycling conditions so I might be of help.

good luck with your pcr.



(((2 mM MgCl2????????)))