I have several COMT SNPs which I designed primers for using BatchPrimer3. I did all my checks and did a BLAST for all primers to ensure it binds to the correct region. All primers have worked fine, except my primer for rs737865 (COMT). For some reason, after running my product on agarose gel, it shows multiple product between 200-400bp. However the expected band is meant to be at 113bp. Why could this be?
Strangely, on SNaPshot, everything works fine. I've run the same sample which shows multiple bands on the gel on SNaPshot, and it gives the correct expected allele calls.
I am not sure why this happening? Why there are multiple bands seen on the gel, when all conditions have been checked.
You can try redesigning the primer using a different reliable primer designing software such as primer premiere. When you do this you may get a different (or similar) primer sequence that works well.
I think that Snapshot is the extension from a primer incorporating a dye so it may be that you have one primer better (more specific) than the other so if the non specific bands are caused by amplification from the other single primer on its own then you might get bands with no detectable dye . Try running a pcr with only single primers and see if you get the unexpected bands
Thanks Abubakar Yakubu Saddeeq , I am actually completing my thesis write up. Our university shut down during level 1 of lockdown in SA due to COVID, so my supervisors recommended I write up with what I have as we were unsure how long lockdown would last. I'm going to still try redesigning the primer
Paul Rutland I had run single plex and gradient PCR for all my primers to ensure they work & to find the optimal Ta. Even then, this particular primer gave me unexpected bands. Your answer makes sense, as my primers designed for SNaPshot are SBE forward primers, which are more specific.
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