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qPCR with Genomic DNA


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#1 klamc

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Posted 22 February 2013 - 10:57 AM

Hi community,

I was wondering if anyone as information about doing qPCR with genomic DNA using SYBRGreen. Can anyone give me information, or refer me to papers? I do qRT PCR to study gene expression, but I not sure how to translate the technique to gDNA, for example how should I dilute the DNA for calculating primer efficiency, or if there are special conditions that are unique to qPCR with gDNA. My objective is to do genotyping.

Thanks.

#2 Multiplexion

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Posted 22 February 2013 - 01:30 PM

One way is to assume that a cell contains 6.6 pg DNA. With this info you can simply translate the DNA concentration in "cell equivalents" per µL.
Next you can adjust your DNA to e.g. 1000 cell equivalents per µL and perform a tenfold dilution series. This dilution series is applied to your PCR in order to determine the detection limit of your assay.
Multiplexion - quality control for your cell culture




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