I ran qPCR using two primers for my target genes in different tissues. I got sharp bands for one primers, but not the other, I had smeared bands. What happened with the cDNA? Degraded RNA could be the cause for this smeared bands? I have attached pics from my gels.
Please share more information, about the picture (by marking each gel) and your qPCR result.
Well about the result, since one of the 2 primers have given good result your template must be good. How about the primers? both have similar Tm and Ta? Did you run gradient PCR to confirm the best annealing temperature? Did u run PCR using both the primer at same Ta? If the 2nd primer has different Ta, then have to run them separately.
I agree with Krzysztof Witcher, they seem like dimer primers, except the first pic from left, the other upper pics don`t seem to have any bands!!!!! if your primers are 25 base , the dimer primers will stand next to 50 bp of ladder !!! so you can distinguish it from your specific bands!!!
I completely agreed that its primer dimer Krzysztof. You should run proper markers with gel too. If there is a problem with bands and smear then decreasing primer, MgCl2 and sometimes dNTPs concentration also helps. If problem exists with primer dimer then there can be two things either primers are not working or your annealing temperature is low.
There is nothing wrong with the template as one of your primer pairs was successful. Normally, smeared bands is a clear indication of a specificity issue and problems with primer design. I would suggest running the PCR rxn once more with increading Tm and if the problem persits you should redesign your primers.
It may also be possible ... your one of the primer is not stable to bind and may bind to differnt reion of DNA. Better to analyze primer sequences.
I will agree with people comment before , different Tm than optimal may couse primer to bind in different region with no nesecerly 100 % homology to your primers, if I were you I will design new pair of primers.
If you test some tips above as run a gradient PCR, it may be a problem with your primers design.
Hi everybody, thanks for your comments. I have a lot of to think about it...and change on my procedure.
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