I'm a tech who's still getting the hang of running PCR. I've been doing it for about 6 months with three different genes (mop3fx, cre, and per2luciferase) and I've noticed that my mop3 primers very rarely give good results. I've been getting very faint bands, or no bands at all, and when I use the same DNA with a different gene I get great bands. I've ordered and re-ordered the primers twice so far. I have a working stock of 10uM and multiple small aliquots to minimize freeze-thawing. Is there maybe some rookie mistake I'm still making?
Here's the sequences for my primers
OL6013F: 5' AAT CAC CTT TTG GGG AGG AC-3'
OL6014: 5' TCA TCA GAG GAA CCA GGG TAA-3'
OL5436R: 5'CCC TGA ACA TGG GAA AGA GA
Any tips or advice would be much appreciated.
Add some PCR additive such as DMSO (2-4%) into the PCR reaction, just in case your specific gene is GC-rich. DMSO and other PCR additive (such as BSA) can increase the specificity and the efficiency of your PCR. This is proven and well documented. Please try one and let us know the result.
Good luck.
Thanks for the response! I've already tried using betaine with no luck, but I'll try DMSO and see how that works.
By the way, I used IDT OligoAnalyzer 3.1 ( https://www.idtdna.com/calc/analyzer ) to check your 3 primers. The Tm for them are around 55oC. What Ta (annealing temperature) did you use?
Yes, try to lower Ta and do a PCR. Do let us know if it works.
Good luck.
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