I am running gel electrophoresis to confirm multiple target genes from PCR (not multiplex). Gene sizes range from 1.4 kb to 1.8 kb and the annealing temperature is 59 C. The same PCR reaction conditions are used for all the genes. I am getting some light bands less than 100 bp. Are these primer multimeres or primer-dimer etc? Can somebody guide me why these bands appear and how to get rid of these?
I think that the small bands are single primer and that you are using too much primer in your pcr. To test what they are set up 3 tubes as if you were running a pcr but do not add dna to the tubes and tubes 2 and 3 only have one primer in each. Run the pcr and load the product on a gel alongside 2 samples of diluted primer only, This will tell you if the bands are primer only ,primer dimer caused by a single primer self annealing or a primer dimer between both primers. To me that image looks like you have both interprimer primer dimer (larger band) and hairpin looped primer.
For your normal pcr use less primer and use a hot start enzyme for better results
Hello Muhammad
I agree with Paul Rutland. In addition to the above I would like to mention a few more points.
1. Try increasing the annealing temperature (use gradient PCR to optimize the annealing temperature).
2. Try using PCR enhancer like DMSO. (Use 1ul DMSO per 25ul PCR reaction).
3. Try to increase the DNA template concentration.
4. As mentioned above try to use hot start PCR because primers tend to have low complementary to each other.
Good Luck.
Yes, agree. Primer dimers. There are online tools for primer analysis, including check for primer dimers of your primers. Example: https://www.sigmaaldrich.com/technical-documents/articles/biology/oligo-evaluator.html
Thanks very much Yuan-Yeu Yau Niren Khomdram Malcolm Nobre Paul Rutland your answers really help.
Your primer concentrations are high and yes they might be primer-dimers or single primers as well.
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