Minimum quantity of DNA per sample to pool?

[yamelttam]

Hello,

I'm preparing ~250 individually barcoded samples (16s metagenomics) that will be pooled in equimolar quantities for a lane of MiSeq.

I am planning to pool 25ng of each sample; however most protocols that I've read use much more than this.

Are there any disadvantages to pooling smaller quantities of PCR product from each sample?

It seems to me that as long as the final pooled library can be submitted at 10nM then all should be ok, but I could use a second opinion.

Thanks for you help!

[nucacidhunter]

10 nM should be fine. For clustering depending on the system up to 2nM is used.

[microgirl123]

I convert my ng/ul concentrations (obtained from Qubit NOT Nanodrop) to nM, then pick the sample with the lowest molarity (unless they are all very high) and dilute all the samples to that in 10 ul.

Then I pool 3 ul of each sample - this gives me pretty even indexing on the MiSeq. Also, the V3 kits like you to start with at least a 4 nM library.

[yamelttam]

Great, thanks for the quick responses!