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 de...

     This week in lab, we began with starting over with our experiment. Last week, our RNA results came out very low and dirty, most likely due to improper handling of samples. For this experiment, we are looking to see if the gene PFS in Deinococcus radiodurans is expressing when put under oxidative stress with hydrogen peroxide.       We started growing our samples on Monday for a total of 24 hours of growth. Tuesday, we started the dilution process. We ensured all 6 samples were individually measured for 1 OD600. After diluting our samples, we the introduced 3 of our 200uL of  D. rad.  in TGY samples to 200 uL of 200mM of H2O2 for 30 minutes. Realistically the H2O2 was at 100mM due to it being diluted once it was added to the samples. For our controls, we just added 200uL of TGY for consistency in volume. Once incubation was completed, the cells for all the samples were washed thoroughly with TGY and pelleted for RNA isolation.  ...

     This week in lab, we continued to isolate RNA from D. rad. The difference is we are actually starting the experiment part of our project. Last week we focus on verifying our primers for our reference genes Sec A and GAP 3. It was determined that because Sec A was 105% efficient and GAP 3 was 97% efficient, we were ready to move on with our experiment. The gene that we are targeting is PFS. PFS gene is a gene that encodes for methylthioadenosine/S-adenosylhomocysteine nucleosidase. Its important to the activated methyl cycle. So we are trying to see if oxidative stress to this gene, effects the AMC in any way.      Our first step was to grow cells. We are continuing to grow D. rad for 24 hours but we changed the volume of TGY we are growing it in to 10mL. After 24 hours of growth, we determine that we wanted to do three controls and three tests with a total volume of 200uL of cells at 1.0 OD600. We diluted it by tube and ended up with the following resu...

                This week in lab, we did RNA isolation on   deinococcus radiodurans.   The reason for doing this is so we could synthesize cDNA. cDNA is a template we are using to study gene expression in   D. rad  when it’s exposed to oxidative stress.   For now, we are doing qPCR for our primers on the gene Sec A to see if it’s a good reference for our experiment.               On Monday,  D. rad  was inoculated into 250mL of TGY media as well as three TGY agar plates. The broth is being used for RNA isolation and the plates for freeze back. On Tuesday, a gram stain was performed on the inoculated TGY media and determined that there was no contamination and RNA isolation was ready to be performed. 3mL of our sample was spun down into a 1mL Eppendorf tube. The process was performed as such:   D. rad  inoculat...

     At the start of this semester, we started with doing a "Biotech Bootcamp." This week of instruction included lab safety, lab protocol, as well as having time to teach/remind students how to pipette and make media/agar. One important protocol we talked about was regarding labeling. We clarified that all media, agar, bacteria, ect. needs to be labeled with the date, contents, initials, volume, concentration, type of bacteria (this is all depending on what the contents are). Labeling also was an issue when it came to waste beakers in the lab. Now all beakers that contain waste must be labeled clarifying whether it was chemical or bacterial waste. We also discussed lab cleanliness. This included washing your hands before and after doing anything in the lab and cleaning off surfaces. Something that was an issue last semester was people leaving powdered media on the scale after measuring, and not wiping things down property. All equipment must be cleaned before and af...