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Old 06-13-2018, 11:14 AM   #6
Location: Baton Rouge, Louisiana

Join Date: Feb 2010
Posts: 31

I haven't yet tried bead based normalization, but I have tried some plate normalization methods like SequalPrep. It did not work well in our hands. For larger sample numbers, like when we are doing 16s, we now use fluoro-plate reader, then create a normalization plate template for our EPMotion. This works best, still get a few outliers but not bad. I randomly check a few samples to make sure unincorporated primers and such are removed.

For smaller projects <24 samples, I just use qubit/bioanalyzer. Calculate and dilute all libraries down to 4nM then pool. I get better read distribution and cluster density using qubit/bioanalyzer than qPCR. I can easliy squeeze out 600million PF reads on our Next-Seq at 85% PF and 85% Q30. My %Phix loading is usually close to being spot-on, so that tells me my quantification is accurate.
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