S4 28: Wow! How Time Flies!

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Wow! How Time Flies!

I still can't believe that winter break is over, and it's almost been two weeks back into the spring semester. This semester I will be continuing my work with Dr. Marshall at ASU-West focusing on Yeast Two Hybrid. I believe that this semester we are going to be testing how different compound concentrations affect the different protein reactions in the Yeast Two Hybrid experiments. I could be a little off as I am still learning about this myself. I am looking forward to getting back in the lab. However, I had to go back to work full-time this semester and am a little nervous on how the semester will turn out. Wish me luck!

S3 27: See Ya Next Semester

See Ya Next Semester

This past week, I focused on testing another an alternative X-gal assay on yeast cells. This was the second alternative procedure that I had tried and it was successful. I had some extra time in the lab before the semester comes to a close, so my research mentor suggested that I try transforming the other two DNA samples again. The rationale for attempting a second transformation was because they previously tested positive for LacZ. Going back to the DNA sample that we were able to extract, I performed a miniprep on the bacteria cells to increase our plasmid yield. Hopefully, I will be able to have a successful DNA plasmid extraction from the other two samples and all three DNA samples can be sent for sequencing.

S3 26: Rounding Out the Semester

Rounding Out the Semester

I can believe the semester is almost over in less than three weeks. I'm a few steps away from getting my DNA plasmid sent out for sequencing. I had a hiccup in the lab last Wednesday. It turns out I used the wrong Transformation Protocol when transforming my DNA plasmids into E-coli cells. This mistake was definitely a reminder to follow my gut and ask questions when I'm not 100% sure of something. I should have known I was making a mistake when I was confused while reading the Yeast Transformation protocol. Luckily the correct protocol was only three simple steps, and I was able to redo the transformation. After the cells were transformed with the plasmid, we replicated the cell colonies onto M63 plates. The purpose of replicating the cell colonies onto the M63 plates is to isolate the prey plasmid from the bait. The colonies that grew on the M63 plates were then re-streaked onto an LB Amp plate to ensure that we will have a sufficient amount of the prey plasmid to be sequenced. Next week I will perform a miniprep on the cell colonies that grew on the LB Amp plate and then they will be ready for sequencing just as the semester comes to a close.

S3 25: Transformation of DNA into E-coli

Transformation of DNA into E-coli

After the X-Gal Assay was performed and we saw the presence of Lac Z, we isolated the DNA from three colonies by performing a Zymoprep for a liquid culture. After the zymoprep was complete, we tested the purity of DNA before we transformed it into E-coli cells. The purpose of transforming the DNA into E-coli cells is to try and isolate the prey from the bait. The transformed cells will be left to incubate for 4 days on LB Amp plates containing Leucine.

S3 24: XGal Assay

XGal Assay

Last week ended with a restreak of yeast colonies onto master plates with minus Tryptophan, Leucine, Histidine with 50mM of 3AT. After letting the plates grow for about a week, we were able to perform an assay on 3 colonies. The assay results revealed that the yeast cells expressed the β-galactosidase enzyme. When X-Gal adheres to the β-galactosidase, a blue pigment will be visible. This activity indicated that there is a LacZ gene present. In order to verify the results, the three samples will be sent out for sequencing. My favorite part about this assay is that it requires the use of Liquid Nitrogen, and let's just say that it was a very fun day of "science-ing" in the lab!

S3 23: Yeast Transformation

Yeast Transformation 

This week in the lab, we successfully transformed the Bait and Prey on minus Tryptophan, Leucine, and Histidine plates with 50mM of 3AT. After a growth period of about a week, each plate was analyzed and sixty-five colonies were restreaked onto minus Tryptophan, Leucine, and Histidine plates with 50mM of 3AT. The plates will be given a growth period of another week. The colonies that are able to grow on these plates will then be assayed and then sent for DNA sequencing. 

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.