Deja Vu Birney's TRAIN Blog

      This week, we discussed how we've been doing our protocol for our AMC oxidative stress project. For this project, we were looking at the expression of the gene Kat A. On our last run, we noticed there was no increase of expression with the gene when exposed to oxidative stress. When reviewing the paper that we've been referencing, we realized that we weren't fully stopping the H2O2 reaction when it was taken out of incubation. This could've cause the affected cells to be killed off completely, showing us no uptick in gene expression for Kat A. We are going to try the experiment one more time by adding catalase to the reaction in hopes of stopping the H2O2 from killing our cells, and instead just stressing them. One other thing we did this week was qPCR analysis. Typically qPCR analysis is done through Excel, but as a hope to understand the math and the meaning behind the numbers, we had a mini lesson on how to do it by hand.     

     For the project, we have been doing an experiment on oxidative stress on different genes in the activated methyl cycle in Deinococcus radiodurans . The next genes were going to be Lux S and KAT A but we decided to only to do KAT A, as that would be the first line of defense against H2O2 exposure. If the D. rad  was exposed to the H2O2, we would see an increase of expression of the gene KAT A.      First, we started with growing our cells. The cells were grown for 24 hours in TGY. Once they were done growing, we diluted the samples to an OD value of 1. This time around, it was decided to dilute the samples by tube rather than as a whole. 1 OD600 +/- 0.2     These are the dilutions we ended up with for our samples. The samples were the exposed too H2O2. Instead of having the cells in TGY during the exposure process, we had T1-T3 in 200uL PBS + 200uL H2O2 and C1-C3 in 400uL PBS. The cells were the put in an incubator at 30 degrees Celsius. After incubation, the cells were put through