I run real time PCR and I find one peak(that is specific for my product) but when I run it in the gel, I saw two bands in gel electrophoresis.
The expected size for my amplicon is 150bp, that is visited in gel, and the other band in gel is 350bp. Can anyone help me what is the problem?
Greetings,
How much extension time are you using for your PCR program? There might be a little concentration of a bigger amplification product because of the primers have homology at some level with a template region (upstream or downstream), outsider your expected amplicon, in combination with using too long extension cycles. I will attach a paper about PCR artifixes and how to avoid them.
Best regards
This may happened for many reason,
1.you may used wrong concentration of primer.
2.your template may have another homologus with primer.
3.Extension time you used.
Dear Maryam Rezaei if you have a good reaction efficiency (more than 90%), side band is not a problem. If you are very interested in solving this question you can clone 350 bpp amplicon and than conduct Sanger sequencing. After that you can affect some reaction components to improve specifity.
The two PCR products can have very similar melt-peaks. What I find concerning is that you are amplifying an unknown PCR product. The qPCR detection algorithm can't tell the difference between amplification of your desired 150bp band and the unexpected 350bp band. So, you can't trust the calculated expression of your gene of interest.
What do you see in your controls?
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