General rules when working with degenerate primers?

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General rules when working with degenerate primers?

Postby mode1111 » Mar 09 2017 2:22 pm

Hi everyone,

As part of my project, I wish to know certain levels of activities of different genes in a very diverse community of bacteria is soil and wastewater. So I extracted my RNA and wanted to do a qPCR. The literature strongly suggests degenerate primers, which is all well and good, except that their annealing temperatures range is wide. I used the same temperatures as suggested by the literature, but I am having difficulty assessing the efficiency of the amplification and the specificity of what was amplified. The melt curve is obviously very "peaky".

Can anyone suggest some advise on this matter? The reason I'm concerned is that I am getting amplification in some sample and in others I don't, and I don't know whether it's a bad experiment or I really don't have anything there.

I am rather new to this. Thank you in advance for your patience.

Alex.
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Re: General rules when working with degenerate primers?

Postby 29yrsExperience » Sep 12 2017 2:58 pm

I know it has been some time since you asked, but the answer is delayed because more information is needed from you. I'm not even sure qPCR is your best approach to the problem. It would help a lot if you gave the citation for the literature you mentioned. Luck!
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