S3 22: Mellow Jello

Mellow Jello
This past week in the lab was pretty mellow. I came into the lab on Monday to take yeast cells that were backdiluated from the previous night and transform them on agar media that included the 3AT. I performed a total of 31 transformations (30 samples and 1 control). The transformation takes about 2-4 days to see any results.I will head back to ASU at the end of the week to check the results.  Additionally, I am working with Dr. Marshall to create a website  with the goal of having the Y2H Transformation protocol and supplemental protocols available so that students from other college campuses like Phoenix College, South Mountain College, and Glendale College can learn about Y2H research and replicate the experiments.

S3:21 Whelp, that's Research.

Whelp, that's Research.....
Whelp, the replica plating didn't turn out as we expected, so we decided to just perform a transformation on a media plate with the 3AT already included. After, I made a new batch of agar media, an overnight culture of yeast cells was started. The next morning, we noticed that the cells weren't growing at the needed rate. We think the cells weren't really growing because of the age of the original yeast culture. Then, it was decided to go back and redo the bait transformation, but I am okay with that because it means more practice for me. The week ended with the bait transformation on a -Tryp media plate. If the transformation is successful, we will see cell growth on the -Tryp plate, and then start an overnight culture in preparation of transforming the cells with the DNA library.

S3:20 Replica Plating

Replica Plating
This week in the lab, I learned a new lab skill called replica plating. Replica plating involves velvet sheets and an apparatus to hold the velvet sheet in place during the replication. Essentially, you take the colonized plate that you want to replica and lightly press it over the velvet and then you take the new plate and lightly place it over the colonies on the velvet sheet, giving you an exact replica of the colonies on a different plate. The purpose of this replica plating was to regrow our colonies on media plates that had 3AT on it.

S3:19 That Yeast is Posionedddd

That Yeast is Posionedddd
This week in the lab, we moved onto the step where we were going to "poison" our yeast cells. When we initially did the first transformation, we noticed that large colonies were forming on the plates which indicated that the bait was still causing transcription and producing histidine. With that observation, we decided that we needed to figure out which concentration of 3AT we could get our cells to grow in without the bait causing the histidine production during transcription. With that said, I made five different concentrated solutions of 3AT and made sample transformations to see which concentration will work best with our needs. Hopefully, the next time I go into the lab, I will be able to see which concentration worked.

S3: 18 Transformation Tuesday

Transformation Tuesday

We performed the yeast transformation of the bait and followed the procedure listed below. The yeast transformation was successful. After the yeast cells were transformed with the bait, we noticed that histidine was still present, so we decided to prepare the cells to be grown on media plates with 3AT. So far in the lab, I have made the media with five different 3AT concentrations. After the media is ready, I will transfer the yeast cells to the media. We will be looking for colonies that are able to produce higher levels of histidine. 

Transformation Procedure: 

For one transformation micro-centrifuge 500mL of competent cells at 500xg for 4

minutes.

Add 0.5mL in EZ 1 solution to wash the pellet. Vortex the cells and discard the supernatant.

Add 50uL of EZ 2 solution and vortex the pellet.

Mix 50uL of competent cells with 3uL of DNA (Bait) and add 500uL of EZ 3 solution and mix.

Incubate samples at 30˚C for 45 minutes.

Sterilize an L-shaped spreader by dipping in ethanol and lighting it on fire but be careful not to heat the spreader.

With an L-shape spreader, spread all the sample on yeast media plate that contains  the –tryp/-Leu

Incubate the plate at 30˚C for 2-4 days so the transformed yeast cells can grow.