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.

S3:17 Hickup in the Lab

https://today.anl.gov/2016/08/safety

Hickup in the Lab
This week in the lab I had a minor setback. On Sunday night a back dilution for the yeast culture was started in preparation for the transformation phase of the project. On Monday morning, when the yeast cells were checked for their phase of growth, it was realized that they were not in the optimal growth phase. After, we decided that it was best to redo the back dilution of cells to make sure they were in their optimal phase of growth before we performed the thirty-five transformations.  I'm looking forward to performing the transformations next week.

S3:16 Transformation Tuesday

Transformation Tuesday

This week in the lab, we took the L40 bait and transformed the cells onto media plates. The cells grew over the course of a few days. Before we transformed the bait onto the plates, I had to prepare the media, autoclave it, and then pour the plates. It is a fun process but can get a bit messy because the agar turns into a jello-like consistency pretty quickly once it gets spilled. Next week in the lab, we will work on the next steps in the transformation process and do a total of thirty transformations. 

S3:15 Falling Back into the Rhythm of Science-ing

First I must express that I am glad the Fall semester has begun. During this semester, I will be at ASU-West under the direction of Dr. Marshall. The goal for the next few months is to become familiar with the Yeast Two Hybrid Protocol. Additionally, I will be experimenting with different Yeast Two Hybrid Library screens. The video above depicts a segment from the DNA Plasmid Maxiprep that we performed this week in the lab. It was as slimy and slippery to work with as it looks in the video! Next week, we will work on transforming the bait. I can't wait!

S2:14 Another Semester in the Books

Another Semester in the Books

The last week of the internship always brings bittersweet feelings. On one end, we are happy to be wrapping up our projects and presentations but on the other end we are preparing to part ways with some of our fellow team members. My favorite part of the semester has been traveling to Vegas with fellow teammates. I learned of another conference happening in DC this fall, and my hope is that I will be able to attend and participate in a poster session. Until then, I will be spending part of the summer learning about extracting bee DNA and helping to test different extraction protocols. The other part of my summer will be spent learning about Yeast 2 Hybrid and associated techniques.