qPCR consistency

[hawaii454-0]

Hello,
We use qPCR (Kapa reagent kits on the Eco Real-time PCR system) to quantitate TruSeq library dilutions. The original libraries are quantified using Qubit fluorometry and Agilent bioanalyser, and a 10nM dilution is made using the Qubit concentration and Agilent peak size. Usually the qPCR quantity is less than the 10nM estimate, and the libraries are adjusted accordingly before clustering. We run phiX controls at equivalent concentrations (i.e. 10pM and 1pM) on the qPCR chip to check the system is working accurately.

Do other qPCR users experience irregularities when quantifying libraries, for example the phiX10 mean quantity being more than 10nM? I regularly get 12nM instead of 10. Can this be trusted?

Also, have you been able to reproduce the same result on the same library on another chip?

Thanks for your help,
Anna.

[pmiguel]

We have taken to running 1:10 dilutions of new tubes of phiX on a high sensitivity chip. Normally instead of 1 nM, we see around 0.8 mM. Nevertheless we treat the phiX concentration as true and use it as our qPCR Kapa kit standard.

However, our most recent tube came out way low -- 0.4 mM. Not sure what is up with that.

--
Phillip