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Old 06-20-2013, 07:13 AM   #3
Junior Member
Location: Washington, DC

Join Date: Jul 2012
Posts: 9

I have been using qPCR to quantify molecules/ul of my amplicons. I have not done a test to see how close Qubit readings are to that of qPCR. I was told by lab members that qPCR is much more sensitive. I typically run ~30 samples on one 454 junior plate and have to do at least 2 qPCR runs to quantify all the samples. I do 3 dilutions for the first run (1:2000, 1:4000, 1:8000) to figure out what dilution I really quantify my samples. This of course depends on PCR cycles and starting DNA amount, but I usually use 1:4000 and 1:8000 dilutions to quantify. BUT I do not use the dilutions to do the emulsion PCR. Most of the time I pipette around 1 ul from each sample, and do one large dilution of that sample. Sometimes 2 ul into 20 ml! I have read and heard from others that they just use Qubit readings and it works fine.
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