I am having one gene whose melting temperature for forward primer is 63 and for reverse its 70. Up to what temperature should I run the annealing temperature?
Until now I did a gradient of 50-60 but no amplification was observed. I am planning to go up to 64 .Can I use them above this?
You can combine the annealing and extension step together in such cases. You can try combined annealing and extension step at 70oC or 72oC . Because taq can give good activity after 68oC.
Yes you can use the higher temperature depending on the AT/GC content of your primers.. the difference between the primers is 7 degree so i would suggest you to go for maximally 72 degree , not more than that, because your one primer is having tm 63, generally people use the melting temp +5 degree than their primer tm.. so better you run a gradient of 64- 72 degree and check where you are getting better result..
Regards
Do gradient from 62-68 you will get the band. Also dNTPs and MgCl2 concentration with respect to size of amplicon expected plays an imp role. There is also a problem with primer design. difference between Melting temp should not be this big. All the best
One good idea is this of Nagesh. In this case you have to check the pcr product. I recommend to do a gradient from 60-75 with at least three different concentrations of MgCl2 (1.5-2-2.5mM). I do it every time with every new primer and I usually have optimum results in one experiment.
I had the same problem.
if the temperature is still too high, you can also reduce the number of oligos to 18bp.
That helps sometimes.
And yes you should really do a gradient
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