I have just finished making some Illumina mRNA libraries using the v2 TruSeq Kit. I used the QUBIT to quantify samples, and got high concentrations (200-600ng/uL). Bioanalyzer traces suggest that there is a lot of material there, as well, and of the appropriate size.
I contrast, I used the Kapa Bio library quantification kit, and got very different numbers concentrations, ranging from .01-90ng/uL. At the end of library prep, after enrichment PCR, the library should be enriched for dsDNA fragments that have adaptors on both ends, and thus are PCR competent. There should not be a lot of dsDNA floating around I don't think..
So, why such big differences?
Attached you'll see a bioanalyzer trace (1:10 dilution) of a library. QUBIT told me concentration was 321ng/uL, but Kapa qPCR says only .12ng/uL..
Can anybody suggest a reason why this may be?
I contrast, I used the Kapa Bio library quantification kit, and got very different numbers concentrations, ranging from .01-90ng/uL. At the end of library prep, after enrichment PCR, the library should be enriched for dsDNA fragments that have adaptors on both ends, and thus are PCR competent. There should not be a lot of dsDNA floating around I don't think..
So, why such big differences?
Attached you'll see a bioanalyzer trace (1:10 dilution) of a library. QUBIT told me concentration was 321ng/uL, but Kapa qPCR says only .12ng/uL..
Can anybody suggest a reason why this may be?
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