Week 7

This week, my labmate and I started to focus more on another project that we started involving Flo11 gene and GFP expression. The main idea is to isolate the Flo11 proteins from two strains of yeast using magnetic tiny beads. The main proponents of this protocol were to lyse the cells and isolate the proteins from the rest of the part of the cells.

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Week 6

During this week, I continued with CRISPR-Cas9 in which I added the European PET111 gene into the migrant strain and vise-versa. Then I left the plates in an incubator and waited for the colonies to grow. After 3.5 days, on a NAT antibiotic plate that was KAN antibiotic resistant, very tiny colonies started to grow up. I swapped three of the colonies and performed a PCR and only one of the colonies worked. The next step is to get the PCR sequenced to verify that the gRNA is actually in the plasmid, which would ensure the CRISPER-Cas9 worked.

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Week 5

In addition to starting my first trial of CRISPR-Cas 9, my advisor introduced a few more projects to me and my labmate. The first project is to test yeast strains and their plastic adherence. For this project, we have started our strains of interest and performed transformations and later we will perform CRISPER-Cas9 to the yeast that have high and low adherence.

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Weeks 3&4

Since I finished performing PCRs on the DNA strains, I have started the CRISPR process. The first step of the protocol is to perform a “pML104 Miniprep.” pML104 is the plasmid that I am using in this CRISPR protocol. The next step is to digest the plasmid, which is essentially the process of cutting the plasmid. Luckily, my labmate found digested plasmids, so I did not have to perform the first two steps.

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Week 2 in the Lab

During the last two weeks, I have finished performing PCRs on the rest of my yeast DNA strains. After a few trials, I got successful PCRs for all of the DNA strains, which were verified by gel electrophoresis. Then I cleaned and concentrated my PCR products to get rid of unnecessary enzymes and other products. After cleaning the PCR products by using a Zymo kit, I used a machine called a NanoDrop to get the concentrations of each of my cleaned PCR products in which most of the concentrations were around 60 ng/microliter, which were ideal. After getting relatively well concentrations, I sent my DNA strains for sequencing (Sanger).

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