Continuing RNA Isolation on Deinococcus Radiodurans When Exposed to Hydrogen Peroxide

     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 @ 200uL                           Control 1- 1.04 OD600 @ 200uL

Test 2- 1.04 OD600 @ 200uL                            Control 2- 1.04 OD600 @ 200uL

Test 3- 1.07 OD600 @ 200uL                            Control 3- 1.04 OD600

    After completing our dilutions, we then added 200uL of 200uM of H2O2 to Test 1-3 and added 200uL of TGY to Control 1-3. The cells were exposed to the H2O2 for 30 minutes at 30 degrees Celsius. We ensured that we flicked the tubes to ensure it was homogenous. After the 30 minute exposure, a wash was performed on both the exposed cells and control cells. The cells were pelleted and the supernatant was removed. TGY was added and cells were reconstituted into the TGY wash and were spun down once more. After getting our final pellet, the supernatant was removed and we continued with RNA isolation. We followed the same protocol as last time.

    When RNA isolation was completed, we then nano dropped for our results. Our results are below:

Test 1-3
Control 1-2

Control 3

    What these results are saying is our samples were not only low in RNA, but also very dirty. Possible errors could've been too many hands on the samples. There were 3 people total working on the samples to there could've been a lot of variation when it came to that. Also, just overall lack of attention to detail within the protocol. As a group, it was decided that there will be a timeline we will go off of every week so only 1 person is working on a portion of the project at a time. Next week, we will regroup and try this new method.