For the amplification of nrfa gene touchdown PCR is preferred. But we are facing problem with it for organism. Is there any other method of PCR for it?
Hi, which Taq are you using? Maybe you should try another Taq! Cheers, Nadine
I had the same problem. Try to elevate the cycle numbers, or use the amplified product as a template for another reaction.
Alternatively, you can do southern and try to find the full length script and isolate it to be used for amplification and in this case I suppose you can lower the annealing Tm. since you have minimized the chance of non-specific amplification.
Good Luck
The specificity of conventional PCRs decreases as the sequence complexity of the template DNA increases. The greater the complexity, the greater is the chance that the primers will bind promiscuously to sequences other than the intended target. The number of mispriming events can be reduced by optimizing the concentrations of the components of the PCR, in particular the concentrations of Mg2, primers, dNTPs, and template. Further minimization of off-target amplification can be obtained by using the most stringent annealing conditions that permit efficient, specific annealing of the oligonucleotide primers to their targets.
Okay, that's a good DNA Polymerase for difficult PCR. I thought you can buy another one but this one is fine.
How do your protocol look like?
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