Hi all,
I did this PCR with my reference and test DNA. I had tested the two primer pairs with my reference DNA and they worked pretty well. I ran the PCR and obtained bands with different band intensities in absence of any primer dimer artefacts. Quality of both DNA's is pretty good. I had also included two positive control plasmids that came along with a kit (which also contain the required primers) and they showed a strong amplification. As a result, after PCR cleanup, I obtained different concentrations of PCR product. Has anyone come across anything like this before?
I have to confess, I do not really understand the question.
You use two primer pairs in one PCR reaction - fine. I do not see the reason, but fine.
There are some imaginable reasons why the bands representing the two products are of different intensity. The most obvious is that the primers do have different binding efficiency with the PCR conditions.
amplicon number vs DNA amount problem:
On the other hand -even if the number of amplicons is the same:
example: 200 amplicons of 2000bp give a signal of double of the intensity of 200 amplicons of 1000bp. why: double the amount of your staining dye can be 'bound' to a sequence that has double the size.
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