I am having trouble amplifying a sequence from cell-free DNA. I tested this primer before with DNA from a cell line, and it worked fine. However, when I tried PCR on human cell-free DNA, nothing got amplified (gel with no band). Specifically, here was my input:
Positive control: 5ng DNA from cell line (fragment size ~ 1-2 kbp)
Test sample: 5ng cell-free DNA (fragment size ~ 160bp)
I am sure the primers work (they have been tested and used before). Does anyone have any suggestions or experience on this?
Hello,
May I know the quality/purity of your DNA sample?
what is the ratio of your DNA 260/280? Good quality DNA will be ~1.8 .
because the quality/purity of DNA samples also effects the primers annealing.
Goodluck :)
Hi there,
You should run an other control to test your DNA is OK for PCR by using an other set of primers for which you know proper product should be obtained. If no product then your DNA prep is not appropriate for PCR, if product is formed then there something wrong specific to your system.
Yes, a too high concentration od template (DNA) may affect PCR efficacy.
Hi again,
About DNA concentration: 5ng of genomic DNA is actually not too much in a PCR mix (for instance recommendation for Phusion polymerase is 50-250ng per 50µL reaction)
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