Rahul Palnitkar, Weeks 1, 2 and 3: Genotyping, Gibson Assembly, and Small Intestines
Note: Weeks 1, 2, and 3 all blend together, and so I decided to make a blog post for all three. Week 2 was my actual start at the lab, and Week 3 was a shorter week, due to the Fourth of July Holiday.
So far, my lab experience has been extremely interesting and fun, if not a bit surprising. I came into this lab thinking I was going to be working with brown fat, as that is the main focus of the lab, so I was quite surprised when I found myself working with intestines instead. Before I came to the lab, my supervisor had found that in mice, whole body deletion of Prdm16, which is required for brown fat development and maintenance, led to greatly reduced intestinal length, killing the mice within seven days of Prdm16 knockout. This phenomenon had not been previously observed, and so the project I'm working on is an entirely brand new area of research.
My first task was to genotype mice and find a particular mouse with Rosa26CreER, an on-off switch that can be activated by tamoxifen, Prdm16 flox/flox, p53 flox/flox, and Casp8 flox/flox. Those last two genes regulate apoptosis, and we want to see if knocking out those two genes will stop apoptosis in the intestine. I genotyped perhaps 40 mice, meaning that I had to run 160 separate PCR reactions. This task wasn't too unfamiliar, as many of the techniques I used—DNA extraction, PCR reactions, and Gel Electrophoresis—I already knew, thanks to Biotech with Mr. Sham.
My next task was to grow some enteroids, which are miniature intestines grown in vitro. To grow these enteroids, we had to dissect some mice and isolate the upper third of their small intestine. This was my first time dissecting an animal, and I thought the whole experience was...well, an experience. I cannot say I'm fond of the smell of mice or intestines, but it was still quite an interesting task. The enteroids themselves came out quite well, and soon we can run tests on them.
My final task last week was to design a plasmid containing a modified version of Prdm16 and transform the DNA into E. coli. First, I extracted the pieces of DNA from gels in a process known as gel purification. Next, we designed and ordered the primers using a program called Geneious. Unfortunately, due to the location of restriction sites, we had to use a more complicated method—Gibson Assembly—to create the plasmid. The plasmid also had an ampicillin resistance gene, so that only the transformed bacteria would survive on the agar plate. However, something went wrong, and all of the bacteria died, meaning that the Gibson Assembly failed.
In addition to lab work, we have lab meetings every Tuesday, and us undergrads (because I am technically considered one) attend a discussion series every Wednesday, which is quite fun. One day, I even got to see someone defend his thesis. It's been quite lively here, despite it being summer. I should also note that the members of my lab are great—especially my supervisor, Rachel. She is a wonderful teacher, and I'm lucky to have her as my supervisor.
As for transportation, I take the train from Trenton to 30th street station, and then walk to the Smilow building from there. The train ride is around 50 minutes, and the walk is usually 15-20 minutes, or more if I'm particularly sleepy in the morning.
So far, my lab experience has been extremely interesting and fun, if not a bit surprising. I came into this lab thinking I was going to be working with brown fat, as that is the main focus of the lab, so I was quite surprised when I found myself working with intestines instead. Before I came to the lab, my supervisor had found that in mice, whole body deletion of Prdm16, which is required for brown fat development and maintenance, led to greatly reduced intestinal length, killing the mice within seven days of Prdm16 knockout. This phenomenon had not been previously observed, and so the project I'm working on is an entirely brand new area of research.
My first task was to genotype mice and find a particular mouse with Rosa26CreER, an on-off switch that can be activated by tamoxifen, Prdm16 flox/flox, p53 flox/flox, and Casp8 flox/flox. Those last two genes regulate apoptosis, and we want to see if knocking out those two genes will stop apoptosis in the intestine. I genotyped perhaps 40 mice, meaning that I had to run 160 separate PCR reactions. This task wasn't too unfamiliar, as many of the techniques I used—DNA extraction, PCR reactions, and Gel Electrophoresis—I already knew, thanks to Biotech with Mr. Sham.
 |
| My workbench during a genotyping procedure |
My final task last week was to design a plasmid containing a modified version of Prdm16 and transform the DNA into E. coli. First, I extracted the pieces of DNA from gels in a process known as gel purification. Next, we designed and ordered the primers using a program called Geneious. Unfortunately, due to the location of restriction sites, we had to use a more complicated method—Gibson Assembly—to create the plasmid. The plasmid also had an ampicillin resistance gene, so that only the transformed bacteria would survive on the agar plate. However, something went wrong, and all of the bacteria died, meaning that the Gibson Assembly failed.
 |
| Gel purification |
As for transportation, I take the train from Trenton to 30th street station, and then walk to the Smilow building from there. The train ride is around 50 minutes, and the walk is usually 15-20 minutes, or more if I'm particularly sleepy in the morning.
Comments
Post a Comment