Hm, yesterday's attempt to do two rounds of enrichment for hypercompetent mutants yielded very few colonies (about 10 total). That's probably because I didn't let the NovR cells grow to a high enough density before transforming them with KanR DNA. There were so few cells present with the KanR DNA was added, 5 x 10^5/ml - 6 x 10^6/ml based on the numbers of colonies that grew on the plain plates after the second round transformation).
But I can do the second round enrichment again, by pooling all the NovR colonies that grew on these plain plates. I have between 1000 and 60,000 colonies to pool, depending on the culture, and the cultures were diluted so much that I don't have to worry that any of these colonies might be KanR. And the colonies are all NovR transformants from the previous round, though many of them are identical descendants of the original mutants.
Plan: Put some BHI on the plates and scrape them to resuspend all the colonies. Dilute the pooled cells in sBHI to OD600 = 0.1, then dilute 50-fold in more sBHI. Grow (37°C, shaking) for 2-3 hr, until OD600 reaches 0.05. Add KanR chromosomal DNA and let continue growing for another hr. Dilute and plate on plain, Nov and Kan plates.
Progress: OK, I've counted the colonies (previously had just estimated them), pooled them (excluding one contaminated plate), and now they're growing in 10 ml sBHI at a starting OD600 of 0.002. Now I need to pour lots of plates.
A new kind of problem
12 hours ago in RRResearch