I am doing methylation specific PCR for various genes. I did bisulphite treatment referring protocol by Herman et al. In my methylation specific PCR result for all the genes ,I am not getting the desired band , but for all the genes , a band near 50 bp is coming. I don't know why is it happening?can any body tell me why is this occuring? and what can I do for its troubleshooting to get the desired product and to avoid this nonspecific band? I did simple PCR to check whether this is a primer dimer, but it was not primer dimer .
the 50bp band is not an aspecific product, they are primer dimers
do you have an alternative amplicon for bisulfite converted DNA? you can use that to check your bisulfite conversion.
if this is ok, and you' re sure your primers should work (if you want to check, bisearch is a free online tool which also allows in silico pcr for bisulfite converted DNA)
if that's all ok, try lowering your primer concentration (I don't know how much you are using but something like 1pmol in a 10ul reaction is usually sufficient, and the less you use the lower the chance of primer dimers)
last but not least, try a different PCR protocol, with higher annealing temp. Personally I have good experience doing first 10 cycles with a decreasing annealing temp, and then 30 cycles of a lower temp. (this way, in the first 10 cycles the annealing temp is relatively high, which doesn't allow primer dimer formation, giving the true pcr product a head-start)
gl,
Arnoud
Thanks to both of you for replying. I have checked for primer dimer by doing PCR with template DNA (without Bisulphite modification) using primers for methylatoion specific allele as well for unmethylated allele, but in that case, I did not get any band. So, I thought It is not primer dimer. If ,in your opinion, it is primer dimer , so why is it coming at the same position for all the genes? I have checked the conc. after bisulphite treatment , it came around 16 ng to 58 ng in different samples? Is there any other method to check for quality of bisulphite treated DNA? I have checked my primers with " Bisearch". There is no problem in primers. I am using 0.4 micro molar primers .I will try with decreasing primer conc. and increasing annealing temperature.
I will try with gradient too. Actually, I picked the primers from some papers and did PCR according to their protocol.So, I think there is no problem of polymerase inactivation. And anyways , taq polymerase is stable at high temperature . so, there should not be any chances of its degradation.
Thank you.
Hi Lalita,
I agree with you that the enzyme degradation is unlikely to be the cause of the problem.
I think the concentration of your bisulfite converted DNA is fine, but this has no information value regarding efficiency of bisulfite conversion.
Good luck with your experiments, I've also tried using somebody elses primers a few times, but never got them to work...
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