Summer Research Report

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Submitted By sakurawolf14
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The first weeks of the summer fellowship in the VDS stream was a bit hectic. They consisted of learning protocols and techniques. Three plates of PNiC-BSa4 colonies were grown by my partner and me that week. These were later used by the majority of lab students who had reached the cloning stage. My first PCR with plasmid pgbr22 failed, but the second was successful. PCR with pmCherry had to be completed 3 times to get a successful result and PNIC-BSa4 PCR had to be completed twice to get a successful result. The three practice PCRs were completed the 5th week of lab. Protein expression using PfDXR was started in week 3 and the sample of protein was purified the following week. I received my target protein Rickettsia prowazskii FabG (RpFabG), responsible for epidemic typhus, during week 5 of research. I worked on designing tail primers for this specific protein with two other researchers in my group that also had the same target. A virtual RE Digest gel was also done with PNiCBSa4 accepting vector and Fab G 3-ketoacyl-ACP reductase insert to help visualize and understand the next steps for our research. Primary PCR was completed with an oligo primer mix for RpFabG, followed by Secondary PCR and PCR2. The sample was nano-dropped after PCR2 to determine that the concentration was high. My first attempt had a concentration average of 9.4ng/uL, so I had to restart and complete primary PCR twice before being able to move onto secondary PCR. The primary and secondary PCR gels showed that the concentration was weak so I made a second oligo-mix after consulting with Dr. B. I completed primary and secondary PCR once again with both the old and new oligo-mixes. With the old oligo-mix, primary and secondary PCR were successful. I added 1.2uL of oligomix instead of 1uL to the master mix and that proved to substantially increase the concentrations that showed up on the gel.…...

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