S-STEM TRAIN Scholar Emerson Turensky

 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....

 Introduction:  The previous week in lab involved a series of plasmid extractions to collect the bank of plasmid we will need to complete mutagenesis. From there, we started preparing the materials called for in the protocol we plan on following for cloning in the pgRNA plasmid vector. We started building our primers for the kanamycin gene we plan to target with PCR to clone into our vector. We reattempted transformation of pwtCas9 as well. This blog will go through how we went about the week and our results that follow.  Methods 1. Grow E. coli with pRHAM on LB plate with kanamycin concentration of 30ug/mL. 2, Grow E. coli with pgRNA on LB plate with Ampicillin concentration of 50ug/mL. 3. Grow E.coli with pwtCas9 on LB plate with Ampicillin concentration of 50ug/mL. 4. After 24-48 hour incubation period, gram stain each plate to confirm bacterial species. 5. Refer to Zyppy Plasmid Miniprep Kit to extract plasmid from each culture after overnight growth. 6. Run plasmid e...

 Introduction: This past summer, the time in lab has been focused around the successful mutagenesis of D. sonorensis. Our preliminary goal is to target the Carotenoid pathway to "turn off" the production of the classic pink pigment, which will result in white D. sonorensis.  We began with a proof of concept trial using pRad1, which went smoothly and showed us transformation can be done with D. sonorensis. Since then, our transformation attempts with the plasmids needed for mutagenesis have posed challenges. We have been unable to confirm any successful transformation with the plasmid responsible for cutting the target gene, pwtCas-9. To confirm transformation in the cell, any particular plasmid will contain antibiotic biomarkers that will gift the cell a new resistance to the specific antibiotic on the given plasmid. The biomarkers found on pwtCas-9 do not cooperate well with the bacteria chosen to transform. The TetR gene has been a poor biomarker, since D. sonorensis itsel...

 Introduction: This past week, the lab analyzed each project and made decisions on what direction to move forward. I was unable to perform any lab work due to not being on any project at the time. While awaiting for an open research opportunity, I decided to try to gain a fuller understanding on how to use some of the common tools found around the lab. I learned the procedure to take proper nanodrop readings.  Methods: 1. With a Kim tech wipe and deionized water, clean the parts of the nano drop that will touch the subject along with the pedestals. 2. Soecify OD values. 3. Pipette 1-2 µL of  blanking solution onto lower pedestal and lower the arm. 4. Tap the blank button and wait for a measurement to be complete. 5. Lift arm and clean both pedestals with a new laboratory wipe. 6. Pipette 1 to 2 µL of simple solution onto the pedestal and lower arm. 7. Start sample measurement by tapping “measure”. 8. When sample measurement is complete, the spectrum and reported values wi...

Introduction:  This week I joined Jacee’s group project, focused on transforming caeni. I saw an oversight of the transformation process, and what procedures need to be done to make sure this process flows smoothly. We also performed a plasmid extraction of pwtCas9. This blog post will provide an oversight of the plasmid extraction process, as well as how we plan to use it.  Methods: 1. First, we pipetted 600 µL of our culture into each culture tube. We had 4 tubes for pwtCas9 and 4 tubes for pdCas9.  2. Centrifuge tube down to pellet and remove remaining broth into bio waste.  3. For each plasmid, two of the tubes are packed twice and two of the tubes are packed only once.  4. Add 100 µL of lysis buffer to all tubes. 5. Inverter either by flipping tube or pressing the stopper of the pipette up and down to get it properly mixed. 6. Add 350 µL of neutralization buffer and invert.  7. Put tubes into the centrifuge for 2 to 4 minutes or until clear. 8. Pipette...

 Introduction: Through this past week, my team and I have been collecting the data needed to complete a full poster surrounding our project. We performed a catalase test on D. sonorensis to wrap up the biochemical tests. In our preparation, certain tests had to be redone to ensure our information was presentable. While the time spent actively performing lab procedure was minimal this week, this blog will outline the methods use as we extrapolate the final biochemical information we could surrounding D. sonorensis and it's plaque.  Methods: Performing a catalase test on D. sonorensis: 1. Prepare a one-way streak plate of D. sonorensis and allow it to incubate for 24-48 hours. 2. Add 2 drops of 3% Hydrogen peroxide to a large colony on the culture dish. 3. Observe for the formation of oxygen gas bubbles.  Observing homogenization differences of D. sonorensis when suspended in TGY versus R2B: 1. Prepare two Eppendorf tubes- one with 3ul R2B, the other with 3ul TGY. 2. Inocul...