I am trying to amplify the promoter region of 2.5 Kb of a rice gene, it is highly GC rich, but I could not amplify it even after changing primers. My primer is totally 43 bp including the 15 bp hang over and restriction enzyme site. I did PCR at different temperatures. Please suggest some solution.
you do not give enough detail of pcr conditions to make a sensible answer but generally I recommend doing the pcr in a mixture with a final concentration of 1Molar betaine. This destroys hydrogen bonding and makes high GC sequences melt more like high AT rich sequences so the template re anneals at a lower temperature so the primers have more time to anneal and extend in the pcr reaction
I agree with both of the previous answers, for us to give more beneficial answer we need to know more. Some other possibilities for your lack of amplification could be poor quality template DNA, your primers may require degenerate bases, could your primers have intra-primer homology or inter-primer homology causing self-dimers or primer-dimers? Another potential problem with your template DNA could be secondary structure formations.
First, I think you should run the gradient PCR. Second, increase the duration of denaturation, annealing because you have mentioned above that promoter region is highly GC rich and fragment size is approximately 2.5 kb. Moreover, use amplifx software to run in silico pcr to analyze the primer quality and reaction.
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