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-   -   Which method is best for DNA quantification? (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/72897-method-best-dna-quantification.html)

mwalt2 04-29-2010 07:12 PM

Which method is best for DNA quantification?
 
I have many samples containing a bacterial genomic DNA. I want to measure the quantity of 7 unique sequences within the genomic DNA and compare them to each other within the same sample. I am thinking that absolute quantification using a standard curve is the best method to use. Due to the number of primer sets (7), setting up a standard curve on each plate would not be possible using all 7 primer sets.

Is it advisable to set up a standard curve for 1 sequence (A1) and then measure the amount of A1 in 26 samples. This would be repeated for sequences A2-A7. Is one able to then compare the amount of A1-A7 sequence in the same sample (S1) across the 7 plates? I think this might be ok since you are no longer comparing Ct's, but you are comparing copy number based off of the standard curves.

Would a relative standard curve or efficiency-corrected delta-Ct be any better? I don't think the relative standard curve would be needed since I am not comparing values across samples, just the number of unique sequence tags within each sample.

Thanks for any advice. I've only used qPCR as a measure of gene expression in the past, so am not as sure how to just quantify tags within the same sample of DNA.


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