Normalization of samples before pooling

[discoveradnan]

Hello everyone!

Can you kindly tell me what is the best way to normalize the samples before pooling? The application I will be working on is screening different diseased genes for mutations.

Thanks

[williamhorne]

I would follow the protocol pertaining to the kit you are using. FOr NextSeq 500 we do Qubit on the libraries and it works beautifully.

William

[Ancestro]

I usually work with qubit quantifications after the PCR cleanup. Because we normalize based on Molarity, the Qubit conc is converted through this page: http://molbiol.edu.ru/eng/scripts/01_07.html.
Normaly, the conversion factor is 1.5 but that differs on the average insertion length.

Then, you must dilute your products to 4, 2 nM to pool them. I do not recomend you to dilute less than 2 nM, and preferably repeat the library prep (or concentrate) before you consider the sample to be pooled.

[kmcarr]

Lately we have been pooling without first normalizing the individual libraries. Equimolar amounts of each library are achieved by adjusting the volume added to the pool and then the whole pool is diluted down to ~10nM (or lower for low concentration libraries). The pool is then qPCR'd prior to loading. Using this method we seem to get better balance in our pools, but I'll admit I don't have hard data to back this up.

[luc]

If the concentrations do not vary wildly, I do not see any purpose in normalizing before pooling. We don't do it.

[LOH]

We use tap station/bioanalyzer to get mean size of library, use Qubit to get conc. of library, then convert it to molar conc. for pooling.

[sajoshi]

I quantify each library with Qubit, in triplicates and take average. Then calculate how much you need for a 70 ng pool (for example). Change the pool concentration if needed to adjust final volume of pool to 15 or more. Then we do Qubit on the pool and dilute 1:2 and do Qubit again. Dilute only those which are >20 on Qubit. Use the 1:2 pool for qPCR. I think qPCR on the pool works better than doing qPCR on each library.