Hello, I am assessing the expression of a miRNA in cells Hep-G2. I am using a stem-loop primer and pulsed RT to make DNA from my miRNA. I am also running agarose gels, at 4%, to visualize my PCR products. As a positive control, I bought a small DNA oligo, which has the same sequence to that of the miRNA I am interested with. When I run the PCR product from the DNA oligo on an agarose gel (4%) I get two bands, one of them at the expected size and the other immediately below of the expected one. I ask your help to understand what it is happening. I should be getting just one band as it is a pure DNA oligo. Thanks!
Easy way out is gel Elute fragments which are according to you the fragment of interest, secondly there is hairpin structure formation which can show two confimations of same fragments,
what sizes are your 2 bands please?
Is it possible that the smaller band is primer dimer or is it a tight narrow band
Thank you Sandeep and Paul for answering my question. Paul, my bands are about 60 nt. The product that I am looking for is approximately 60 nt long. The unexpected band is immediately below. Sandeep, I agree with you, likely it is a secondary conformation of the fragment. Would it be fair if I run an agarose gel at 2.5%?
if this is a conformational problem you should consider denaturing PAGE or possibly running the agarose gel at higher voltage so higher temperature which might force the product into a single conformation,Because the bands are small running hotter will cause more diffuse bands which is unfortunate but might prove the point so I would prefer PAG
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