Summer Research Report

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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…show more content…
The PCR2 sample from the previous week was purified through PCR clean up and nano-dropped. The concentration was 68ng/ul. The concentration of this sample was low (<100ng/ul) because I had skipped the first step in the cleanup process that prepares the column filter for maximum DNA binding. PCR2 and PCR clean up were completed again and the concentration of the sample was averaged to be 147.9ng/ul. Since the concentrations of the samples were adequate, I moved onto PNiC-BSa4 cloning and prepared a sample to be DNA sequenced. RE digest was completed on two PNIcBSa4 prep samples, with the gel indicating that both samples were successfully cut and nano-drop results indicating high purity levels with low concentrations (~32ng/uL). Results from sequencing were nucleotide blasted and confirmed to be PNiCBSa4. I talked to a mentor about my samples and was told that I probably needed a higher concentration ≥100ng/uL, for cloning to be successful. In order to increase the concentration 4 samples of 50uL each were cut and PCR clean up into one sample that had a concentration of 123.5ng/ul. The cloning protocol was completed that week and when the plated were checked, no colonies were present on either of the two plates. My first attempt at cloning failed. The process was completed again, and the sample concentration was averaged to be 112.1ng/ul. The second time, only one
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