Hi there,
I have a problem to get a conclusion in my PCR results. I have nested-PCR product in which the first run size is what i expected to be. However, the second run is ~100nt larger than what it has to be. Please help in to resolve this problem.
*note: the primers are obtained from WHO's recommendation for genotyping of Rotavirus
Regards
Hello Nazanin,
Are you getting two bands in the second PRC or one nonspecific band?
It seems that longer band is a nonspecific product which is usually due to non-optimized PCR condition.
Could you give the sequence of primers you have used ?
Dear Nazanin,
firstly , check your sample by using a new specific primer, if it gave positive band the problems were take in PCR optimazation, So, be careful, and I give you my article about Rota virus as attach , I designed new primer for Rota virus , it is very active and easy procedure
regards
Dear Nazanin,
check the annealing temp. if possible make gradient PCR to get the optimal temp. some nested PCR gave me less size 100nt. in influenza protocols. if the size nested PCR product is suitable for sequence. I recommend to do that
Hi Nazanin,
I hope you know the sensitivity of PCR technique. It is not easy to determine when and where non-specific amplification occurs without observing cycling condition, amplicon size (for both round of PCR) and agarose gel document. Since the primer sequences are selected from WHO material, theoretically it hold good specificity without internal complementary sequences. However, please check the sheet (give while primer synthesis) and re-verify it.
If everything is right, please read further to have some idea. First, it is a nested PCR. Hence, there is a possibility to have completely non-specific or incomplete band of target which may not be in the agarose gel in the first round of PCR itself. If such condition occurs then there is chances to obtain larger band in second round of PCR. Otherwise, there is no way to have larger amplicon (technically beyond the length between two primer sequence ). Try you to use slightly higher annealing temperature or used touch down PCR approach to fix the problem easily.
Hi Nazanin,
Have you a copy of the sequence of nucleotides in the first PCR and its product and located the primers on it for the second? When you have established this then titration of reagents and annealing temperatures is required for optimum conditions in a gradual and step by step process. PCR conditions can vary so much depending on the day. Short cuts to optimise the PCR often mean they take longer to work. You may wish to design your own primers when you have the sequence in front of you. First principles are always a good place to start.
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