Week 1

The first week I was given a couple articles to read through that explained more about the specific project I would be working on. I learned that the enzyme telomerase has four domains: the N-terminus domain (TEN), telomerase RNA binding domain (TRBD), the reverse transcriptase domain (RT), and the C terminus extension domain (CTE). My project would be ascertaining how the RNA component in telomerase is binding to TRBD. In order to this, I would be isolating the TRBD protein from telomerase and expressing it in bacterial cells. I would then be purifying the TRBD protein in an attempt to determine a crystal configuration of TRBD. 

In order to begin my project, I first had to learn about the techniques used to express proteins. I learned how to prepare protein and dna gel stock solutions and how to use an SDS Page instrument to isolate protein based on size. I also learned how to use a Kodak imager to visualize the size of protein I have. 

After observing others in the lab, the first task my mentor had me do by myself was a 'mini-prep'- a technique to isolate DNA from all other protein in the given sample. The mini prep I did was not specific to my project, I did it to practice the techniques I would be using for my project. My mentor needed only plasmid DNA in his sample so he could use that plasmid DNA as a vector or a means of inserting in a foreign DNA fragment for cloning a specific gene. In order to do this, I followed a protocol my lab had laid out which told me to add specific buffer reagents to first wash the the cells in the sample several times, break open the cells, and add neutralization buffer to keep the DNA inside the cells intact. 

After the mini prep, I used a nano drop to measure the concentration of DNA to protein. I first blanked the instrument with 1 microliter of water, then inserted one microliter of my sample to obtain an absorbance reading of my sample which I could use to calculate the concentration. Because I was supposed to be isolating DNA, the concentration of DNA was expected to be much higher than concentration of protein. Because the aromatic rings in DNA absorb light at 260 nanometers and the amino acids in protein absorb light at 280 nanometers, my sample should have absorbed significantly greater amount of light at 260nm than 280nm. 

 

This was the absorbance vs. wavelength graph of my two samples, and in this you can see that the mini prep I conducted was successful because there is a significantly higher absorbance at the wavelength DNA absorbs than the wavelength of light at which protein absorbs. However, it was unclear why one of my samples had a higher absorbance at 260 nm than the other sample because both the samples were supposed to be the same.

After obtaining these results, my mentor had me run the DNA sample on a gel so we can detect the size of DNA we had. Below are the band sizes of DNA I was able to visualize through a Kodak imager. The first column depicts the ladder I inserted in the first well of the gel which is supposed to be a marker or a reference to compare the size of my samples to. The two columns to the right of the ladder show the band sizes of my two samples. It was unusual that the Kodak imager displayed two bands instead of just one band and the sizes of the DNA samples seemed a little off. My mentor said that we would need to perform further experiments next week on this sample to see what went wrong and why the imager displayed two bands instead of one.