PCR inhibitors in DNA?

[grace-ac]

Just ran a qPCR plate targeting V. pectenicida in DNA samples.

The filter sample qPCR plates have been going great, and today was the first time I added in eelgrass swab DNA into the wells.

The efficiency of the run was 130% which google says can mean that there are PCR inhibitors in the templates.

I'm thinking that this is likely only because of the eelgrass swab DNA (the 5 plates I've done of just filter DNA have all had efficiencies 90-110%, which is ideal).

Any thoughts on what I should do? Melanie Prentice mentioned I might want to try a clean up kit on the DNA to get rid of inhibitors.

[grace-ac]

A note - I ran a few swab DNA samples on the nano drop over the weekend and fairly low DNA ng /ul (see post here)... so it's likely not that I have too much DNA loaded in the plate??

[sr320]

a couple of tests

Dilution test. Take 3–4 eelgrass extracts with decent signal, run neat / 1:5 / 1:10. A clean sample shifts ~3.32 Cq per 10× dilution. If the shift is smaller than that, or the 1:10 gives a better-than-expected Cq, you have inhibition.

Spike test. Add a fixed amount of your standard into an eelgrass extract and into water. ΔCq > ~0.5–1 cycle = inhibition, and it works even on samples that are negative for the target.