- S. cerevisiae
So my overnight culture grew, that’s cool. The E coli did not though. That’s not. To make matters worse, the -80C freezer had some weird malfunction and it was nearly RT in there. Hopefully nothing is ruined. I’ll restart experiments on Monday and make new batches of yeast and e coli spreads and liquid media as well. Then I’ll make some glycerol stocks so that I can be ready for all eternity.
On top of all that I’ll reacquaint myself with doing transformations and purifying plasmids because there are some vectors that I need to replenish. Not for the purposes of these experiments, but just because.
I’ll try and do some preplanning over the weekend and through Monday.
We streaked two plates and poured a bunch of stock into the third plate (hence the whole lotta cells). I didn’t do the streaking because they weren’t my cells.
I did pick a colony from the third plate and streaked that onto a new plate, so hopefully there will be good spreading.
I parafilmed the plates and stuck them in the fridge.
Onward to tomorrow.
The yeast has spread into colonies (yay), but the E. coli have not. I don’t get what is happening there, but I’ll try growing them in liquid media and seeing what happens. Tomorrow I’ll check on the e. coli and decide what to do from there. In the mean time, I’ll also start a liquid media batch of yeast to grow side-by-side with the e. coli.
Let’s get it…
and poop to some extent…
Ahem! I started cultures of e. coli and s. cerevisiae streaking cells on some plates to be incubated overnight and beyond. From here I will pick colonies and grow flasks of them to get familiar with the growth rates so I can take data. If all goes according to plan, I’ll be getting some preliminary results by the end of the week!
Here is some info about my cells:
E. coli: XL1-Blue MRF’ competent cells that are designed to replicate pBluescript
Yeast: S. cerevisiae of the variety g160/2d
Another page for project outlining that will change over time.
UPDATE: I changed the embed code so the map has live update capabilities.
I spoke with Kelly Trujillo about cell synchronizing after Koch had mentioned to me that we may need this for ddw yeast studies. He mentioned that he was going to be synchronizing for a study that he was setting up and that I could come learn the process. So I took him up on his offer.
I have to say, the protocol is surprisingly simple. Kelly grew a colony, diluted it to about 0.1 OD and then let it divide up to 0.4 OD. We looked at the asynchronous growth and then he added some mating pheromone (details to come) to some of the culture to halt the cells in G1 phase of cell division. From here, he could synchronize them to enter S phase by using hydroxyurea (HU) after washing out the mating pheromone. The remaining culture was arrested in G2/M phase with the drug Nocodazole.
We paused during each step to look at each culture (asynch, G1, S, and G2) but I have no pictures. I do have the knowledge to do it myself and hope to incorporate this in the DDW experiments shortly. A preplanning thread will come soon.