yeastfeaster

[yeastfeaster]

this whole blog is just probably gonna be for me ranting or sharing cool shit that I do in the lab!

3/6/24

today I did a plate assay to get a quick (3 point) anisotropy based binding curve for Pol alpha on fluorescein labeled template/primer DNA. a couple months ago we did a whole bunch of these with different conditions and they were working great, but now that we're back to doing them, they aren't working anymore. I just did a quick 3 point one today (0nM, 6nM, 150nM Pol alpha) to test and see if we're getting the same mP values we were getting before, and we are not. I think something is up with our plate reader cause the values were like 10x less than they should be at our previously discovered binding saturation (which started around 150nM pol). big sad for today in that regard.

Another thing I did today was restreak a yeast clone I've been growing. I transformed it a little bit ago, screened a couple colonies, and now I'm restreaking a few of the clones positive for my DNA. The DNA in question is a linearized plasmid integration, which I integrated in the middle of the yeast Trp1 operon. The DNA also has N-Terminally STREP-tagged Sen1 flanked by a Gal promoter so we can overexpress it. I tried linearizing my plasmid with 2 different restriction enzymes in the Trp1 operon this time (being SnaB1 and BSU361), because the last time we did this linearization, some weird stuff happened. our lab's yeast strain acts weird with Trp1 integrations if you linearize with BSU361, and we had done this before. The resulting protein was inactive and during the prep we could see a whole bunch of strange truncated product. We think relinearizing and reintegrating might help that.

Anyway, I'm confirming stable integration of Trp1 by streaking onto YPD (regular yeast food) plates, and then back onto -W (no tryptophan) selection plates. Our yeast can't grow on -w without Trp integration, so growing them on that plate confirms they have what I'm looking for. Additionally once I'm done restreaking I'll do another heat-pop PCR screen to check for Gal1-Sen1. if the PCR is able to amplify that region, it confirms that the plasmid got integrated into the yeast.

I'm doing a whole bunch of other stuff right now but I think im gonna provide background for that when it actually comes up. ill share some pics too! here's the first restreak of that clone onto YPD. I took a colony from each of these guys and streaked them onto -w earlier today.

Also, here's that colony screen i was talking about. most of the clones had the integration, seeing as there are bands of a size that I expected in each one (except 5s its dumb)

[yeastfeaster]

Revised Mechanism of Hydroxyurea Induced Cell Cycle Arrest and an Improved Alternative

BioRxiv: http://dlvr.it/T3cq4M