Category: Ava H – Molecular Biology

Today was a shorter day. I previously made a miniprep culture to isolate plasmid from E.Coli. I drew 10mL LB Broth into 4 test tubes and drew up a colony and dipped it into a test tube. Repeated 4 times. Overnight, the prepared culture amplifies the E.Coli and DNA to be harvested. So, today, I resuspended the culture using a centrifuge, four different buffers for each sample, and supernatant. By the end, I moved the sample to a microcentrifuge tube and drained/isolated the DNA to the bottom.  Then my time in the lab was cut short before a friend gave me a tour of UNC.

My lab technician showed me protein separation and detection used in gene therapy. Proteins can be separated by size with SDS PAGE (polyacrylamide) gel and stacking gel, which I made. Western blots are used to detect proteins, which uses protein-specific antibodies to visualize protein bands. We went to a room and pipetted chemiluminescence over the surface of a western blot paper and placed it in a machine. The B-actin (in all proteins) showed a bold line of fragments.

^Ellie’s membrane with protein samples (not visible) after adding primary and secondary antibodies

I continued my digest, starting with “part 2”, using KPnI-HF, which incubates at a lower temperature. After incubating, my lab technician helped at CIP (calf-intestinal alkaline) to the backbone, which removes phosphate groups from the ends of the strand, keeping the split ends separate and the insert “sticky”. Then I used electrophoresis to isolate the strands I want and ligated them. The last step was electroporation (same as Day 1). The last step would be to use a miniprep screen or sequencing to confirm change but I didn’t do that.

I started a new digest. Before I used HSS11 and Eagl Hr enzymes to cut the plasmid. Through the software, I selected Pcil and BspHI enzymes. After cutting the plasmids I blunted the ends with Klenow and dNTPs and began PCR (polymerase chain reaction) purification to isolate the pure DNA strands. I am attempting this digest on my own, although my lab technician still teaches me temperature moderation is important throughout the process. For example, enzymes must be stored at below-freezing temperatures, but incubated at 37C.

I started by purifying a ligated DNA specimen from my running digest experiment. Then, I used electroporation to transfer the DNA samples into ecoli cells and placed them in a warm room to amplify. Then, my research technician taught me how to use cell imaging/screening to determine if cells are contaminated by a virus. Finally, I was taught how CRISPR edits CF-infected DNA and how to use a sequence editing software to simulate which enzymes could cut DNA sequences in largely different places to make cut DNA strands easy to compare. My next step is to make sure the DNA specimen properly amplified and test my chosen enzyme through a new digest.

Ava will continue work she started over the summer with molecular biologists at UNC. She will use this space to explain her work and post images of some cool lab stuff.

UNC Gene Therapy Center