I have a constant issue of background in my assay. i am thinking its primer degradation, so how can i check that.
Do you mean primer degradation before using it in the assay or caused by the assay conditions please?
Sometimes primers are deporinated, which is lead to smear in your gel. It can be resolve by resolving stock primers with TE buffer.
The PCR background is not a reason that the primers are degraded. First, why should your primers be degraded? The degradation of primers is a slow process and requires special conditions and great efforts. Second, the background of PCR, what are you meaning here?
I believe that you have a classic overamplification, a lot of DNA, a lot of everything and especially of PCR cycles.
i mean primer degradation after resuspension.i have uploaded the image plz have look and give me suggestion. i feel i am getting this background due to primer degradation.Before applying this assay on real samples i optimized this assay.
as I say above, you have a classic overamplification, a lot of DNA, a lot of everything and especially of PCR cycles.
Reduce the number of PCR cycles 2-4.
it looks to me as if your biggest problem is a primer dimer amplification ( the smallest band). Getting rid of this may make your amplifications better. Use a hot start taq, less primer and a negative dna control and run the gel at a lower voltage and for longer to resolve the bands better
What exactly will be the consequences if the primer is degraded and how do we confirm that it's the primer which got degraded.?
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