I am trying to amplify intronic region of miR-1017 from Drosophila genomic DNA. Have done this several times before with no issue. Currently I am screening flies for mutation and for the purpose I am trying to PCR amplify the region. But the same working conditions isn't working anymore. I have tried with fresh primers, different set of primers, proteinase K treatment of template. It works for few round and then I start getting blank gel picture only with the ladder.
Any suggestions on this?
Greetings,
I don't mean to simplify your problem, however, have you double checked your PCR protocol condition? More than once, I had to re-set the original running settings because of someone unintentionally changed them while handling the thermocycler.
Good luck
I would check the quality of your DNA. Has it undergone numerous freeze-thaw cycles? If it's feasible, I would reperform a DNA isolation, the primers do not seem to be an issue from your troubleshooting.
If the DNA can only be amplified for a few round, maybe you need to consider the possibility of contamination in the DNA. Check the quality of DNA (concentration and purity) and run it on gel to see the integrity.
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