Progress towards the tweezers experiments

On Wednesday I did a second test of ways to attach cells to coverslips.

This time I had pre-cleaned the coverslips by soaking them overnight in acid-ethanol (1% HCl 70% ethanol), and then air-dried them. I spotted solutions of poly-L-lysine on two of these; one goth 20 µl of the 0.01% solution recommended for dipping coverslips into, and one got 20 µl of the 0.1% stock solution. I let the spots sit for 5-10 minutes and then removed most of the liquid with a pipetman, and left the coverslips to air-dry. Then I spotted a bit of dense B. subtilis cell culture onto each coverslip. overlapping the spot of poly-L-lysine. I left the cells on the coverslip for about 5 minutes, and then rinsed each coverslip 5 times 1 second in a large volume of saline. Results: many many cells were stuck on each coverslip in the area that had been coated with poly-L-lysine. Both concentrations worked equally well.

Next I'll try using more dilute solutions, and try dipping coverslips in the solution, with and without rubbing until they're dry,as one experienced user suggested. I'll use the new competent cells I've just made. And I'll test whether the styrene beads (with and without DNA) stick to the treated coverslips, and whether the beads stick to the competent cells.

I also had one slide that had been incubated in sealed plastic tube with a small volume of the HDMS (?) silane, which is very volatile and should deposit vapour on the coverslip. I took several other coverslips and spotted HDMS on them and let it dry, and then spotted the cell culture on, and washed them as I did with the poly-L-lysine coverslips. Results: No cells stuck at all.

Today I'm making more B. subtilis cells competent. This time I'm freezing some of them, so I don't have to go through the competence ritual every time I want to test anything. And I'm also transforming them with my new prep of DNA, hoping to get a higher transformation frequency. And I'm plating some of the transformants on minimal plates with no casamino acids, with and without methionine, so I'll be able to score the frequencies of Met+ and Met+ Trp+ transformants, and thus calculate the fraction of the cells that are competent. (I'll need to wait 2 days for the colonies on these plates to get big enough to count.)

On Wednesday I also visited my physics collaborators. I attended their lab meeting - I think we'll start using their format on alternate weeks, with everyone given a short time (5-10 minutes) to update the others on their progress. The other weeks we'll continue with the one-person-presents format. And I had a good discussion with the PI, so now I know more about the practical issues. And she gave me a couple of reviews, which I haven't started reading yet.

Got to go DNase-treat and plate my transformants...