Dear all research
I'm have the problem about picking the melting temperatures(Tm) of the primer. Different of formula or program give the different Tm of the primer. Which program or formula is giving appropriate Tm, your favorite one, to determine your designed primers.
Thank you
Using different polymerase requires different calculation of melting temperature. For instance, the melting temperatures calculated in Primer3 may be good for Taq, but they may be too low for Phusion Polymerase. I would suggest you to check the protocol of the PCR enzymes you are using, and see what formula they recommend.
I actually prefer designing primers "by hand". I use the very simple formula where a C or a G is 4 degrees while a T or A is 2 degrees. I try to keep the primers as close possible to 60 degrees in total. 10 G/C and 10 T/A (or as close as you can get) is preferable. When running the PCR I usually go down 2-4 degrees from the calculated melting temperature. Works very well for me.
You can also go to the UCSC Genome Browser. They have a function called In silico PCR where you enter you primers and you get a list of what the primers will amplify and the melting temperature of the primers.
Using different polymerase requires different calculation of melting temperature. For instance, the melting temperatures calculated in Primer3 may be good for Taq, but they may be too low for Phusion Polymerase. I would suggest you to check the protocol of the PCR enzymes you are using, and see what formula they recommend.
In general, the formula Tm= (wA+xT) * 2 + (yG+zC) * 4 works well for small nucleotides, with less than 17 nucleotides.
*w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.
For longer sequences is preferable to use a formula that takes into consideration both the G+C content and the length of the primer, such as:
Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC), or
Tm= 60.81 + [0.41*%(G+C)]-500/n
*n is the number of nucleotides of the primer.
These formulas are calculated based on specific salt concentrations and pH, but they use to work well in my case, when I use Taq polymerases. With Fusion Polymerases I prefer the calculations based on the thermodynamic parameters of Breslauer et al., 1986, which are the ones that you can find at the Primer3 webpage.
Dear Ditthawat,
if you want to calculate the melting temperature of your primers you could use http://www.basic.northwestern.edu/biotools/oligocalc.html. It's easy to use, gives you GC-values and various options to choose your conditions. I usually prefer melting temperature around 55-58 °C and a GC% between 40-60%, but that differs from Primer to Primer and the used template DNA.
I hope this answers helps,
Best wishes,
Philip
I use primer blast, in the thermal cycler with gradient I put seven tubes with differences between 0.5°C, one with sugested Tm, 3 up and 3 down
I use Unafold
http://mfold.rna.albany.edu/?q=DINAMelt/Homodimer-simulations
It gives all information with melting simulation.
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