Hello
I ran a Gradient-PCR, annealing Temp ranging from 63 to 67.
I am getting a faint band at the top of the gel and down to my GOI band. (Image attached).
I have tried nearly everything from Primer dilution, Temp dilution, decreasing annealing timing, polymerase enzyme dilution, and Fresh Template. But the results are the same every time. So any suggestion on what else I can Try?
Non-specific bands appearing at high annealing temperatures (Tm) in PCR reactions can be caused by a variety of factors, including primer-dimer formation, genomic DNA contamination, and mispriming. Here are a few suggestions to help you troubleshoot and resolve this issue:
By implementing these strategies, you should be able to reduce non-specific amplification in your PCR reactions and improve the specificity of your results.
These videos might be helpful to learn about Gell electrophoresis trouble shooting https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
Nikolay Klyashtornyy Thank You So much for your reply. Did you see the gel image? I did the Gradient up to 67. I used an Invitrogen kit to isolate the genomic DNA and checked its purity, I have tried the different PCR conditions (Reduced Taq Pol conc, Primer Dilution, Template Dilution and etc). But top bands are still coming. my target size is ~1kb and non-specific bands are coming above that. How?
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