- Make frozen stocks of the retransformed Topo + KaiABC (original strain)
- Make a 20% glycerol mix (40% volume of 50% glycerol, 60% volume of culture), a little under 2 mL total volume
- Miniprep the rest
- Ligate the purified PCR product from PCR extraction #5. There are 4 reactions: LC1PUR1 3kb, LC1PUR1 400b, LC2PUR1 3kb, LC2PUR1 400b
- Transform the ligated Topo + KaiABC and Topo + 400b with Top10 competent cells
Our 4C fridge door was open when I came in. There was a big puddle of something leaking out the bottom, which I cleaned up with about eight pounds of paper towels.
Frozen stocks of TP
Yesterday we inoculated 4 liquid cultures of the "TP" strain of Topo + KaiABC, from our first transformation on 2006-7-11. As a reminder, this is the same strain we sent out for sequencing on Wednesday, and the same strain we used in our first couple PCR mutagenesis attempts (1, 2) and our diagnostic digest.
I made frozen stocks of each of the 4 cultures, in a 20% glycerol mix:
- 400 µL 50% glycerol
- 600 µL liquid culture
Minipreps of TP
I miniprepped the remainder of the 4 liquid TP cultures. I eluted with 30 µL H2O. The miniprepped DNA is in our freezer in the tray labeled "Templates".
The cultures are in the -80C freezer along with our other frozen stocks. They are labeled TP #1-4.
Ligation and transformation of LC1PUR1 and LC2PUR1 3kb segments
Yesterday we gel-purified the 3kb and 400b bands from our LC1PUR1 and LC2PUR1 PCR reactions (see the gel image here). Today I found out that we ran out of LB Carb plates in the lab. This means I couldn't transform and plate the positive control that comes with the Zero Blunt Topo Cloning Kit. You may recall that we accidentally left this kit out overnight at room temperature a couple nights ago, so a positive control would have been very helpful to see if the kit still worked.
Since I was unable to make a positive control, and Alain ordered new Topo kits on Friday, and we don't need the 400b bands immediately, I decided to Topo-clone and transform only the 3kb bands from LC1PUR1 and LC2PUR1. My reasoning is that, if the ligation and transformation succeed, we can immediately start trying PCR mutagenesis on the new Topo + 3kb colonies, so it's worth giving it a shot. If not, we shouldn't be too bummed, because we can try again with new Topo next week. Either way, we don't need the 400b band just yet, so it's best not to waste the effort now in case our Topo kit went bad. Also, I'd like to discuss the 400b band with the rest of the cyano team.
I made 3 ligations and transformations: LC1PUR1 3kb, LC2PUR1 3kb, and a negative control with H2O. For the Topo ligation, I mixed 4 µL of each with 1 µL salt solution and 1 µL Topo vector. For the transformation, I mixed each Topo ligation with 40 µL competent cells (normally I wouldn't have use so much, but I took two tubes of competent cells from the freezer, anticipating that I would do a positive control and the 400b bands as well).Each of the Topo + 3kb transformants was plated 3 times and the negative control was plated once. They should be grown up by tomorrow.