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DNA and RNA contamination in RT PCR water controls


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#1 maya

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Posted 17 November 2009 - 10:20 AM

Hi, I was doing RT PCR and got a similar reading of DNA and RNA using nanodrop in my water controls as in my samples (using Progema RT ImPromII kit, two steps). I then repeated both step one and step two separately with just the nuclease free water and from the kit (no samples) and got the same results with nanodrop as before, but when I nanodropped the nuclease free water by itself I got no DNA or RNA. I also nanodropped the RT enzyme and the RT inhibitor by themselves and got a reading of DNA, but much lower than the concentration I got in my water controls. I used a water blank each time so I don't know if that was the right blank for the RT enzyme and inhibitor. Any ideas as to what went wrong? Thanks.

#2 piemmea

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Posted 17 November 2009 - 03:58 PM

Hi, I'm not sure if I understood correctly, but with "regular blank" do you mean that it contains dNTPs? Because in that case, their presence may have biased your reading. Spectrophotometers do not discriminate between dsDNA, ssDNA, RNA or nucleotides (or anything else that absorbs at its wavelength). Do you see your products when ypu run the gel?

#3 Adrian K

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Posted 17 November 2009 - 07:14 PM

Dear Maya,
Pls check and troubleshoot your primers, dntps, polymerases, nuclease free water etc to check whether there is any genomic contamination. I suspect something to do with your master mix rather than the water.

Hope this helps.
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