Hi Adam,

1. Yes, we use the standard Illumina protocol for denaturing/loading. The BioAnalyzer gives us numbers to adjust our starting pool to 4 nM.

2. Yes, we denature 5 µl of the 4nM pool.

3. We pool before we quantify, so we just take 5 µl of the pool. In your case, you'll need to pool libraries if you haven't done so. I'd recommend doing it in larger volumes than 0.25 µl though to avoid pipetting inaccuracy. If you've already normalized all the libraries @ 4 nM, then just take a microliter of each to make a 4nM pool, then take 5 µl out from that pool. (You should have plenty of extra library.)

4. We use the NEB Ultra II FS kit starting with 400 ng of input DNA. But other factors can determine final yield as well. For example, I think we only do 3 PCR cycles during the PCR step, but more cycles will get you more library in the end. Some libraries we've prepped are > 200 nM, others are only 5-10 nM. As long as you're above 4 nM, you should be fine.

5. Yes, we add between 3 and 6 µls of the phiX control before loading the sample.

6. Don't think so!

Good luck,
–Dan