This has been quite a busy (and rewarding!) week in the lab. The gBlocks (artificially created double stranded DNA fragments) I was waiting for to construct the Ect2 mutant finally came in the mail. I started creating the mutant by using the gBlocks (which have the mutations in the Ect2 binding sites present in the sequence) as primers for PCR amplification of wild type Ect2. This allows me to introduce the mutations I want in the Ect2 sequence. Naturally fluorescent jellyfish under a dissecting microscope. These jellyfish are actually quite small and some individuals naturally fluoresce under a green laser. The left is just the green laser, the right is both normal transmitted light as well as the laser (which is why you can see some green residue towards the center). In preparation for looking at my Ect2 mutant, I injected some sea star oocytes with other mutated versions of Ect2 to provide a basis for comparison. In addition to T808A or Snowflake, a mutant I have previously posted a video of, I also injected a mutant named Silent Spring (because of the presence of ‘DDT’ in its amino acid sequence), as well as a similar mutant named Snow Leopard. These mutants both interfere with binding sites for a protein called Cdk1, a cell cycle regulator which phosphorylates Ect2. On the Rock front, we received the sequences from the attempted S. purpuratus Rock amplification. For the most part, the sequences looked really clean. We successfully cloned both the front and back ends of the Rock gene into the appropriate vector. The next step is to amplify the missing middle section and combine the middle section with the front and tail to create a complete version of the gene. Additionally, we now have the Patiria miniata putative Rock sequence. After designing primers for the P. miniata Rock, Kara managed to PCR amplify the gene on the first try! This means that I could potentially make another version of the probe using the putative P. miniata Rock, which is exciting!
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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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