What melting temperature to use - (Mar/10/2010 )
Hi! Can anyone help me on what melting temperature, Tm to use for my ISSR primers? I am confused as to what melting temperature to use to determine the annealing temperatures (Ta) for my set of primers. In one article which used the same primers the Ta used range from 52-53.5 degress (depending on the primers) but doesn`t have an explanation how they were determined. Another article which used almost the same primers but for different species, the Ta was fixed to 52 degrees. The primers I bought came with specific Tm, which are way too low if I compute Tm using the formula Tm= 2(A+T) + 4(G+C). And if I compute the Ta (Ta=Tm - 5) using the Tm indicated in the primer labels, what I get are Ta values ranging from 36-54 degrees where most of the primers falling below 50 degrees.
Which options would be better for me:
1) Use the 52-53.5 (primer dependent) Ta;
2) Follow the article with fixed Ta which is 52 degress; or
3) Determine the optimum Ta for each primers by conducting a PCR run using a Gradient PCR machine (which will require much of my time and use considerable amount of reagents)?
Or if you have better suggestions they are most welcome.
If I were you, option 3 is my choice. Also, try reduce your volume to 15ul for your gradient will save your reagent spending problem.
Just my 2 cent. Hope this helps.