Fun with cloning!

Hello All!!

 

So, I arrived in Williamsburg on June 3rd and promptly started lab research on June 4th. Time really does fly when you are fully engrossed in a research project.  Just as a re-cap on my project. I am performing a Yeast Two-Hybrid Assay, to find proteins that interact with the protein, UBE3A. I could go into every nitty gritty detail of each step of the process, but I feel as if I would completely bore you. So, I am going to explain it in a simplified way. A scientific experiment in a wet lab takes many many many steps. Each step takes much longer than what would be expected, especially when working with yeast.

Before beginning my two-hybrid screen, I first had to make my “bait” and “prey”, which are both transcription factors. My bait is E3a and my prey is the human cDNA library. In order to tell if the bait and prey have a true interaction, the bait must be transformed into a vector containing the binding domain and the prey must be transformed into a vector that contains the activation domain.

The first step in my project was to order my gene of interest. My gene of interest came from a lab in California. My gene of interest was bought inside a vector. Once the bacteria swab containing the vector that contained my gene arrived, I had to take a wooden stick and plate out the single colony I isolated on an lb carb plate. I had to wait 2 days for this to grow, then had to inoculate the colony over night, so that I would have enough cells. In order to extract the DNA out of the vector, I used polymerase chain reaction. PCRing it will also add “overhangings” (nucleotides homologous to a vector containing an activating domain or binding domain) to my gene, so that my gene of interest will be able to homologously recombine into the vector. After the 1st pcr, I ran out a small portion of the product on a dna gel to ensure that the dna had been replicated. Then had to clean my PCR product, so that I could just isolate the dna.

I ran a re-PCR to add more nucleotides to the overhangings, to ensure that my gene of interest is inserted into the activation domain. After the re-pcr was completed, I had to clean my PCR product, so that I could just isolate the dna.

At this point, I finally made my gene of interest and it has the overhangings I want on it, so that it can be homologously recombined into the plasmid. I used a restriction enzyme to digest my plasmid at cut sites, so that the gene can be inserted into it.

 

And as all experiments with DNA, I had to run the linear plasmid out on a gel, to ensure that the gene was inserted. Inserting the gene of interest into the plasmid: Perform a yeast transformation. I then extracted a plasmid (w/ gene in it) from yeast. Once I isolated the plasmid extraction, I electroporated it into e. coli cells.

I cleaned the cells and did a double restriction digest, and from the digest, I ran a DNA gel and I saw that my vector and gene of interest were successfully transformed.

Whew deep breath- That is a simplified version of what you need to do for creating a clone. It is hard work, but it is well worth it, because in the end, others can use it and I can as well. I then transformed my binding domain and gene of interest  in the specific yeast strain that I can use for a two-hybrid assay. In my next blog entry I will go into more detail on this.

Highlights of Research: Being surrounded by such interesting and competent individuals. I just started in Dr. Kerscher’s lab this summer, but everyone else in the lab has been working with him for quite some time. They are all extremely intelligent. I really enjoy being able to talk to these people about genetics and molecular biology!

Every Friday we have a lab meeting, where people in the lab present their research. I have thoroughly enjoyed these meetings, because I get to learn so much!