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Old 07-29-2013, 03:08 PM   #1
RezaFalsafi
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Location: Vancouver

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Default Quantification method-iLlumina libraries

Hello, I prepare libraries for RNAseq for a GAIIx we have. I use both High sensitivity DNA chip and KAPA library quant kit to quantify my samples. However, I have noticed sometimes for some samples the concentrations are way off as when I put even 4 pm i do nnot get anything and it seems over clustered(too many clusters/mm2)
any suggestion or any kits/method I could reliably use so it does not have the limitation of not quantifying very highly concentrated samples?

Thanks a lot in advance
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Old 07-30-2013, 01:01 AM   #2
SS00
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For high concentrated samples (e.g. with >5cycles PCR) I tend to nanodrop the sample first to get a rough reading of the concentration and dilute down to a range I have confidence that qPCR can quantify, for me that is 30nM (by Nanodrop).

You might also try swapping out your standards from KAPA standards to one that is more similar to your sample. E.g. if you are qPCRing RNAseq libraries, choose a sample you have gotten good concentrations from at a given loading concentration and use that to prepare your standards for qPCR.
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Old 07-30-2013, 09:45 AM   #3
RezaFalsafi
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Thanks a lot for the help will do so next time.
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