At lunch today I sat beside someone who's been using Illumina sequencing to characterize the genetic and arrangement diversity that arises in a bacterial culture during growth from a single cell. He had lots of practical advice about our recombinome project. Most importantly, because diversity will have arisen in our recipient cells during the growth they do before we transform them, we need to make a DNA prep of a no-DNA added control part of the culture at the same time as we make a prep of the DNA-added transformant pool at the same time, and sequence this control DNA along with our transformed-cell DNA. And when we sequence our donor DNA, we should use the same DNA prep that we use for the transformation.
Other issues: Yeast extract contains yeast DNA that persists in culture media and can come through the DNA prep and contaminate the Illumina library. We grow our H. influenzae cells in 'brain heart infusion'. I think this contains yeast extract, but it may also contain DNA from the hearts and minds of cows. Illumina may have a protocol for removing DNA from culture media, or we could just DNase-treat it before we autoclave it (I wonder how much DNase, for how long...).
Concentration of DNA in the input sample is critical. There's a new way to use PCR to very accurately measure very low DNA concentrations, called 'Digital PCR'; it uses a version of limiting dilution where only some wells contain a DNA molecule.
We had a wonderful reception at the Monterey Bay Aquarium on Wednesday night. But we only got to see part of the aquarium, so now I'm going back to see the rest.