This week, I continued my pursuit of the elusive Rock gene. I am trying two approaches to tag and follow Rock: the first is to use a Rock probe originally designed for Xenopus laevis (African clawed frog), and the other is to design an echinoderm-specific Rock probe. An X-ray of my model organism, Patiria miniata. Photo credit to Edward Nichols and the Hanson-Meekins Animal Hospital. While struggling with the PCR of the echinoderm Rock sequence, I tried injecting sea star oocytes with various concentrations of frog Rock. Unfortunately, this also presented several obstacles; since Rock is a fairly large protein (about 4 kilo base pairs) and maybe easily degraded, it is very tricky to actually see its activity within the cell. However, I did get one successful movie of Rock making waves on the cell surface that appear to overlap and immediately follow Rho waves. Although the Rock fluorescence is fairly dim, the movie is long enough to create a kymograph to further analyze wave activity. Additionally, I am continuing to try to alter probe concentrations and length of time after injection to try to catch more Rock activity. A female (red rock?) crab with extruding eggs bravely caught by Richard. After many unsuccessful runs, we finally obtained PCR product for the full-length Rock gene, as well as the front and back halves separately (thanks to Kara and her perseverance!). After purifying the PCR product, we went ahead with the next steps in making the GFP probe. First, we ligated the PCR product with a GFP vector, thus introducing the PCR product onto a circular plasmid. We then transformed the plasmids into E. coli cells and conducted colony PCR to confirm the transformation success. Outside of the lab, the REU interns all went camping this weekend at Sunset Bay. After being woken up by sea lions barking, we all went tide pooling in the morning. We were rewarded for waking up for the low tide; we found some giant crabs, sea lemons, huge sea urchin colonies, and even spotted the arms of an octopus as it drifted by.
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AuthorI am a rising junior at Carleton College, majoring in biology with minors in Neuroscience and Russian. I'm very excited to be working in Dr. George von Dassow's lab this summer, where I will be studying cell biology and embryology of marine invertebrates. Archives
August 2019
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