I have when doing methylation of DNA kit. when I do the negative control by not adding in the anti-5-methylcytosine antibody, then PCR, I am getting unspecific binding on my gel result, when there should be no binding at all. I have done the protocol several times following it carefully, I am not sure what the problem is. Additionally, when I run my primers with the 5-mC modified DNA, not for the negative control, I am still getting unspecificity by getting multiple bands in my gels. What am I doing wrong?
I think this is because of the non specific binding of your primer to the target sequence. Check your primer sequence with your target sequence to see if there is any possibility of non specific binding. Also during designing a primer one should be very careful to fulfill the criteria of being a good primer. From my experience, primer should have a length of 21-22 bp, melting temp should be 55 to 60C (melting temp of forward and reverse primer should try to keep same), GC content should be >45% and also should try to keep the starting and ending nucleotide G or C, Best of luck
I share the comment given by Tanvir. In addition, you need to minimise risk of contamination as much as possible during performing your procedure. Try to use all possible and appropriate controls to identify the root cause of your problem.
It is not because of nonspecific binding to the target sequence. In genomic DNA my primers are very specific with one clear band. All my primers are about 200bps. I don't think the problem is with the primers. I have done the protocol several times each time being very careful to not have contamination. Is there anything else that could possibly be causing this? I can not figure it out.
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