Hi,
Recently I synthesized a new primer (from a published journal) to be used for qpcr analysis. However, if I want to do a run test to validate the primer, how do I do so using a conventional PCR? Is doing a test in positive control correct? Thank you.
Yes, you can perform a test PCR before going for qPCR. You can use the same template and run the product on an agarose gel. Keep in mind the product length to analyse the results once the gel has been run.
Hello Nurul Kamal
Even though you have chosen a primer from a published paper, I would suggest you run a gradient PCR first to optimize the annealing temperature. Once you get the optimized annealing temperature then run a conventional PCR with positive control as well as a negative control.
I also suggest you to do a gradient temperature for your primer annealing. Then based on your chosen temperature you can run you PCR. In addition you should read read the about the size of band you have to get after your PCR.
Hello. Thanks for the helpful advice!
I ran my first test run today and used the same PCR protocol from the referred paper. I wonder if I could refer to the protocol even though that's for qpcr? I replaced the reagent for real-time to conventional PCR mastermix.
I didn't get any band so I wonder if that was the reason.
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