S-STEM TRAIN Scholar Emerson Turensky

 Introduction This past week in lab, we continued the mission we set out on. We intend to clone pgRNA with kanamycin, to produce a guide plasmid for ours's, and the rest of the lab's endeavors, one that works well with deinococcus. We troubleshoot restriction enzymes and start getting our stock of kanamycin up. We also reattempt pwtCas9 transformation, in preparation for the overarching mutagenesis project that this guide plasmid will fit into. These events went as followed:  Methods PWTCas9 Transformation  1. Perform transformation of D. sonorensis with pwtCas9 plasmid following transformation protocol. 2. After 24-48 hour incubation, inoculate onto plates of both TGY and TGY dosed with ampicillin. 3. Incubate at 30C for 24-48 hours. 4. Observe results and passage successful transformation growth onto TGY plates with ampicillin. Restriction Enzyme Digest 1. Use XBAI restriction enzyme to digest pgRNA following given restriction enzyme protocols. 2. Incubate at 37C o...

 Introduction: The previous week in lab, our group worked on getting a good bank of our plasmids going, as we start beginning the process of mutagenesis. This past week, we started work with the restriction enzymes chosen based off the restriction sites of the plasmid, where we intend to insert KanaycinR gene to allow pgRNA to work as a viable guide-RNA for not only our group, but for those in the lab working towards their own mutagenesis endeavors. The ligation protocol called for gel excised and purified fragments to be prepared before the ligation steps. This blog post will go through how this was done, and what changes were made to yield more success.  Methods Gel Excision and Purification 1.Load sample into gel. Chose a volume with large molecular mass to yield most product through the excision and cleanup process. Expect around half of product to be lost.    2. Locate target band and complete process referring to Thermo Scientific GeneJet Gel Extraction Kit. 3....