i am facing a problem that previously in touch down and in gradient PCR of specific gene, specific band with non specific product come. but after 2-3 weeks when this same gene PCR was performed for optimization to minimize the non specific product with same primers and DNA no product was appear. what is the reason for that.
If the gel tracks are completely clear then one of the reagents in your pcr mix is prbably failing. Use someone elses pcr reagents and if the pcr still fails then the problem is probably with your primers failing due to nuclease activity or depurination. Buy new primers and dissolve them in TE not water.
If there is just a smear over a wide size range in the sample wells then you have pcr contamination and that is a completely different problem. Can we see a picture of the gel please?
There could be several reasons for this.
1) Might be the fragment sizes of your actual amplicon and the one which is non-specific.
2) Primers could have Introns (maybe make sure, how you design your primes for the gene, at gDNA level, or only CDS?).
3) Try a gradient PCR to get your specific annealing temperature.
4) Try alternative PCR reagents.
Hi Madeeha Tariq
The PCR troubleshooting guide (following link) can be helpful to resolve the problem.
https://international.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide
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