Two weeks ago, I run some PCRs, sometimes I cannot get amplification but other times I indeed get it. But now that I am trying to replicate it is not possible, that could not be unspecific bands becase they showed a mutation that I was expecting but no nothind is amplifying. I prepare new stocks of primers, and I have been using everything new. I also isolate new DNA but this is not working, do you have any suggestion?
I've had this issue before. The easiest thing to do is design new primers. Sometimes, fighting the optimization process isn't worth it.
If you are really stuck on using these primers, I would set up a reaction gradient with really low melt temp to a high melt temp. Some machines can do this gradient for you. Probably a spread from 50-62C (assuming a melt temp around 60C). Cast a wide net and see what you get.
Another thing I would recommend is running a positive DNA control. If you are working in a plasmid or a genome, chances are you have some old primers in the lab for a sequence unrelated to what you are looking at (these also have the nice advantage of being known to work in previous experiments). Set up a control reaction to verify you have good-quality DNA and that your polymerase is still working.
That sort of inconsistent result can also mean you are using too much DNA or the DNA has PCR-inhibiting compounds. I know it sound counter-intuitive, but try diluting the DNA 1:10.
And yes, include a positive control.
It sounds like you are dealing with a tricky case of inconsistent PCR results, which can be really frustrating. Since you already tried fresh primers, reagents, and DNA, I would suggest double-checking your DNA quality, sometimes even new extractions have inhibitors that mess with amplification. Also, revisit your primer design and try a gradient PCR to fine-tune the annealing temperature. Make sure your polymerase and dNTPs are still active, and confirm your thermal cycler is running at the correct temperatures.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning