PCR with DNA at different concentrations

[Silvia_84]

Hi all,

 

I was wondering if you can illuminate me or give me some opinions on what can go wrong during my PCR reactions. I extracted bacterial DNA and I diluited my DNA samples 1:10 and 1:100. I tried to run different PCRs to identify a gene. I run a PCR by using the extracted DNA as it is, another one with the DNA diluited 1: 10 and another one with DNA diluited 1:100. I was able to see the product if the DNA was dilutied 1:10 and 1: 100, but not if I used the extracted DNA as it is. I thought that maybe something with DNA extracted during the PCR reaction can go wrong because the DNA is too concentrated, since in the other two cases (when DNA is diluited) it works ...does it make sense for you?Or do you have other suggestions? I tried to run again these same PCRs, same conditions but this time I am able to see the product with the DNA extracted and when it is diluited 1:100 but not if it is diluited 1:10...Do you suggest something?What would it mean?

 

Thank you in advance

[hypervalent_iodine]

High concentrations of DNA are known to inhibit PCR. There are a few possible reasons that I can think of for why. The first is that your primers may be binding to other sites on the template strand. High concentrations of DNA with the same primer concentration would mean that more of your primers are binding to sites other than your target region, and you either aren't getting amplification, or you're getting amplification of the wrong bits. If concentration is super high, the diffusion of Taq through the tube could also be blocked, making the whole thing inefficient. I think that DNA is also an inhibitor of polymerase after a certain point. Could be wrong on that, but I recall hearing it somewhere once upon a time.

[CharonY]

High DNA concentrations also scavenge Mg2+ ions and make them unavailable for the polymerase. In addition, the extract could contain inhibiting factors.