Deja Vu Birney's TRAIN Blog

     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.      RNA isolation and cDNA synthesis took place on We

     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 results. Test 1- 1.04 OD600 @ 200

                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  inoculated in TGY media -Add 1 mL of inoculated TGY to Eppendorf tube, spin down for 1 min. -Remove supernatant without disturbing pellet. -Repeat this process 3x total               Afte