I am working with a N20 mer (20 mer random region) aptamer library with a total length of 56 bps. I am facing the problem of higher sized ds DNA PCR products forming in my SELEX cycles. The size of the ds products appear to be 76 bps in a 4% agarose gel. Is my whole pool converted to a higher sized products?
Kindly help.
Hi, I think you should have optimize the number of PCR cycles to get the desired product. Over amplification may lead to higher sized ds DNA.
Good luck !
I have optimized the cycles and amplification conditions for my DNA library. If I use lesser number of cycles than the optimised one, the positive control and my DNA bands from the SELEX round donot appear at all in the gel.
I will try using qPCR for further optimisation.
Thank you everyone.
If the qPCR doesn't work you could try less cycles or DNA concentration and use PAGE gels for visualisation of your PCR amplification. I don't know how many cycles you are using but for me 24 cycles were more than enough and I could easily see the DNA in agarose gels.
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