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qPCR DNA concentration problem...

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qPCR DNA concentration problem...

Postby s206038950 » Thu Jan 20, 2011 2:52 pm

Bacterial community DNA has been extracted from human stool samples. DNA quantification platform using fluorescence implemented to determine total DNA concentration in extracted sample (bacterial and human DNA). The concentration determined by the DNA quantifier produces concentration results which are 2X more ( 10 ng/ul ) than that which is required for the subsequent qPCR reaction ( 5ng/ ul req.) and YET when I dilute the samples even 1:30, my concentrations are still too high, producing Ct values which match that of the positive control. Contamination has been ruled out due to standard curve dilutions of the positive control producing optimal results. Any help and ideas would be greatly appreciated. Thanks!!!!
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Postby canalon » Thu Jan 20, 2011 4:16 pm

Contaminants in your DNA prep that change your quantification (fluorescence quenching or something like that)?
Patrick

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