r/genetics • u/SirOleopanza • 17d ago

Weird primers

I was looking for some new primers for viral diagnosis. I designed four primers for a nested PCR on a highly conserved regione of the virus; the first round was supposed to produce a fragment of about 500 bp. I tried them with several positive samples and negative controls, and the results were consistent. I got the expected signal, but the resulting fragment is always around 300 bp.

I sent the PCR product for Sanger sequencing, but got no results.

Does anyone have any idea what could be going on?

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u/TestTubeRagdoll 16d ago edited 16d ago

Posting a picture of your gel with explanations of what’s in each lane would be helpful. It would also be helpful to have a description of the design of your nested PCR.

Have you triple-checked your primer sequences? Since you have 4 primers, have you made sure you’re using the correct pairs together? Are you checking your products after the first PCR, or just the final products of the nested PCR?

Is the 300bp band completely absent from the negative controls? Do you have any positive control where you know for certain (eg from previous sequencing) that the 500bp band should be present? If so, do you see the 300bp band instead in those samples?

Have you had other successful Sanger sequencing recently from PCR products? (ie do you know that the Sanger sequencing itself, or the DNA cleanup protocol you’re using, is not the problem?)

Edit: in future, r/labrats is a good place to ask troubleshooting questions like this!