Hi, I ran a standard curve on several DNA samples to determine the efficiency of PCR. However, I noticed a strange thing: the first three concentrations worked quite well, while the last two (least concentrated) DNA samples had Ct values much lower than the sample with the highest concentration. I can't imagine that this is a problem with this particular primer pair; however, I can't think of any other reason for this strange behavior either. If you have any idea what may explain this I will appreciate it very much. Thank you. Pavlina
It would be interesting to know the od260/280 of the template solution. You may have pcr inhibitors remaining in your template dna so as the inhibitors get diluted more the amplification improves. One such inhibitor could be EDTA which removes Mg from solution and the Taq enzyme needs Mg to work
Hey Pavlina. did you make serial dilution for your (DNA) template? If you did that, you are sure they all are 10-fold diluted. then the problem is most likely in your extraction. repeat extraction and include internal positive control with any other target to make sure no PCR inhibitors are present.
you may have to check your dilution ratio, as you need to dilute your to ensure that no inhibitor is present in your sample.
Hey Palvina
How were the Ct value are below 10?
I think you should recheck the concerntrations and dilution for clarity.
What were the Ct values like? It could be that you are diluting something out that was causing higher Cts in your first few dilutions.
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