I bought a primer pair for conventional PCR, the sequence and Tm of both are:
- GCA CCA TGT TTT GAG GTT G Tm 50.7 C
- CGA CTA CTG CTT AGC ATA Tm 40.3 C
By checking the Tm of these primers, it was found to be completely different from the company (bioner inc.) calculated Tm. Moreover, there is a huge difference between the two primers Tm which makes it impossible to perform PCR. Does anyone have any recommendations? I have attached the paper for PCR protocol which uses the same primers and an annealing temperature of 55 C
Sorry..and your problem is???
Depending on which software you use to calculate Tm, results can differ. Also you have to put into account that Mg conc, dNTPs conc andDNA conc affect Tm values, so for every reaction is different. This is why for every reaction (also with published primer) must be done a optimization step
Did you try to make the PCR using the same parameters of the published paper? If you have the same Taq, same dNTPs, etc...you can try with the same annealing temp. Also if you do not have exactly the same reagents, try it, and depending on the results you'll have to optimize Tm, Mg and primer conc...
Good luck!
thanks a lot for your kind reply. I am using Green master mix (fermentas). I think you r right because technical support recommended increasing the annealing temperature of PCR because of the higher Mgcl2 concentration in the published article than that of the master mix I am using. additionally, she recommended primer conc. of 20 picomol. please advice.
Doaa...I suggest you use for the primer, the condition recommended by the tech supp. For the Tm, try to do it at 60, and when you have the results, check if needed to be optimized..but make a test..Good Luck
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