Hi
I did a PCR with three samples and one negative control. However in the negative there is a clear band but below 100 bp while in the other lanes with the three samples the bands are around 300 bp. I know that this can be primer dimers but in the other three lanes I also see very faint bands below 100 bp but also lower than the band in the negative control. It can not be that the primer dimers in the PCR reactions with the three samples are smaller than in the negative control? Does anyone have an explanation for this?
- You often get a stronger primer dimer in negative controls as the primer and enzyme has nothing else to do. When the amplification is going well in the samples the concentration of primer is slowly reduced by incorporation into the product so there is less pd when there is good amplification.conversely when you have much pd there is often very little good template amplification
As Paul Rutland already said, it most likely primer dimer. try to minimize the amount of primers added to the reactions or dilute your primer stocks.
I also already thought about this but in the three lanes with the samples I can also see some faint bands below 100 bp but these are also positioned lower than the possible primer dimer band in the negative control. The length of the primer dimers can not differ between the samples and the negative control?
can we see a picture please?
smaller than primer dimer sounds wrong.....only primer should be smaller than a dimer so possibly your small band is the wrong thing amplifying and the even smaller bands are primer or dimer
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