S-STEM Scholar Alex Hugenberg-Cox Blog

      This week we went back to our own oxidative stress experiment. After a long discussion about our lack of results when using Hydrogen peroxide as a stressor, we decided to switch our stressor from that to Sodium Hypochlorite (NaOCl). Even though we never got to test our results from the exposure where we introduced catalase, Chad was certain they would yield results no different than any of the tests we had done before. Instead it was decided to go with Sodium  Hypochlorite, from bleach.      Before we could start our actual experiment, we would need to see what percentage of sodium hypochlorite would be ideal. We would be using agar plates, and mixing the cells with different dilutions of NaOCl and inoculating. The plates would than incubate for 48 hours before establishing how each dilution percentage effected the cells. We started by doing a dilution from household bleach at roughly 8% sodium hypochlorite, diluting down...

      This week was very uneventful. Chad had put our project on hold for the week, and we instead spent our time helping Lani both with final poster prep and the initial stages of the DNA extraction project. While they had started the project over the summer, the samples they had were in the wrong elution and would need to be redone.      We spent the week doing graham stains, inoculating broths and plates, making gels and refilling more pipette tip boxes than one would thing possible.      All in all we did a lot of busy work that to us wasn't much but helped others when they were to busy to do it themselves.      The conference itself also went well. This was the Arizona Nevada Academy of Science conference. There was a wide variety of posters that were all very interesting and covered a wide array of topics. Two of the ones I spent a good deal of time at was a poster that studied bite forces of sharks compare...

       This week we ran our RNA samples from last week, so i got to make and run a RNA gel for the first time.      Monday Deja transformed a small amount of our T1 RNA sample into cDNA using a new kit. Our usual kit was gone, so in the mean time we tried a small amount of our sample with a new kit that had different steps to see if it would work. The only way to see if it had worked was to run qPCR, so that's what we did Tuesday. Unfortunately it did not work, so we would need to wait for our usual kit to get it, but we still had the rest of the samples.      Wednesday I taught Lizzie and Iditi, to people in the lab who had never done the RNA isolation procedure, how to do it. It was good for me to sit and run through all the steps, and explain each one and make sure it was being done correctly and precisely. It helped me gain a better understanding of the procedure, and what different steps did.      Thursday was ma...

      After reviewing the original paper our project was based on at the end of last week, we decided to try and edit our hydrogen peroxide exposure protocols in order to get a better result. This mostly meant adding catalase at the end of our incubation period, along with some other minor changes.      We started the week of with some inoculations and graham stains, and spent a good amount of time preparing for a biotech program booth that we would be setting up later that day.      Wednesday we finally got to the new hydrogen peroxide exposure. We started by diluting down 11.5M H2O2 down to 200mM. We also diluted down our six groups of cells all down to an OD close to 1.  After pelleting all of our cells, we removed the supernatant, and resuspended all of the cells in 200μl of PBS.  We than re-pelleted, and added 200μl of H2O2 to our test cells, and 200μl of PBS to our control cells. Next we incubated the cells for ...

      This week we discussed the math behind our qPCR analysis, and learned the steps to do it ourselves. While we do get the calculations from a computer, its always good to be able to double check the calculations, and to be able to find where an error is if their is one. We covered the calculations for relative expression (called the livah mathod), not absolute expression as that's the calculations used in our project and also used for most qPCR calculations.       After covering how to do the calculations, we also discussed and reviewed our procedure. We hadn't been getting no significant change in gene expression when under oxidative stress from H2O2. Even when tracking the KatA gene, which should have light up with H2O2, we saw no significant change. When looking at the paper we were basing our procedures on, we saw that they actually halted the H2O2 reaction by adding catalase. After a small discussion, we decided that next week w...

  Last week we decide against continuing with pfs and instead using both LuxS and KatA. While we didn't get to qPCR this week, I did get to perform RNA isolation by myself and and do a new procedure: alcohol precipitate.  We started the week on Monday with a graham stain. All our cells looked good, and we had a ton of D.rad to work with.  Running the RNA isolation on my own was difficult. There was alot of back and forth, and i was constantly leaving and re-entering the hood. Deja was able to arrive in time to help me for the last few steps, but for the first hour and a half I was doing what we usually have two people do. Since we do the RNA isolation in the biological hood, I was very worried about contamination or even just forgetting a step trying to do everything.  I did have a hiccup during one of the steps. I had been given instructions to try to do everything possible in the -20 ice box, instead of keeping it in normal ice when possible but doing normal steps ...

This week we were finally able to do qPCR. We didn't have lab Monday, so we started Tuesday with diluting our samples to prep for RNA isolation. We tried to get as close as possible to an OD600 of 1. After a bit of work, we ended up with the closest dilutions prier to RNA isolation that we've had so far.  Before Dilutions: T1  2.86 T2  2.14 T3  2.73 C1  3.06 C2  3.20 C3  3.11   After Dilutions: T1  0.99 T2  1.01 T3  0.99 C1  0.98 C2  1.01 C3  0.99 Wednesday we did RNA isolation and cDNA synthesis. After RNA our samples we nanodropped the samples to see our concentration.         ng/ μl       A260/280   A260/230 T1  6.6         1.83            1.08 T2  16.3       1.97            1.08 T3  32.6       2.06        ...