Week 2:
Carver College of Medicine

June 3, 2014

This week we focused on projects 2 and 4.

Project 2: We made mini preps for the para ligations (insertion of a DNA fragment into a vector). After plasmid (DNA molecule that is separated from chromosomal DNA within a cell) purification, we digested the six samples using BamHI, which is a double cutter enzyme (it cuts the vector at the insert and outside of it). We cut the vector and when we ran the samples on the agarose gel, we were supposed to see a band at about 7.8kb and one at 4.1kb. None of the samples had the correct bands (when we ran the samples in an agarose gel), so we’ll have to keep working on it probably next week.

Project 4: We also made some mini preps for the RXC clones (where an arginine was substituted for a cysteine at positions 4, 5 or 6 on domain 4 of protein Nav 1.4). We also had to prepare more PCR reactions for the wild type (with I1303C mutation) because when we previously ran a gel with that sample, no bands were found, suggesting that the mutation I1303C didn’t work. We digested the wild type plus the other RXC samples, and ran them in a gel. Since we were trying to see if we succeeded in adding the mutation I1303C into the RXC samples, we expected them to cut just like the wild type when we ran the gel. We had to prepare multiple gels before we got some samples that worked (three colonies, one from each sample – R4C, R5C, R6C). We’ll keep working on this project next week.

It’s been really fun working in the lab and I’ve learned so much about gene mutation and expression since I started working here. What is really funny is that before we start working on projects like these, we (or at least, myself) have such a simplistic idea of what mutating a gene is. I guess I just always assumed we’d insert a mutation into the DNA and that was it. It turns out that the reason people spend their entire lives working on the same projects is because science is way more complex than we think. Take my experience, for instance, we’ve run so many gels and tried so many different approaches to try and mutate these ion channels, and so far we haven’t had much success.