BioTech Lab Entry 7

 Introduction

    This week, a UV test was performed on Deinococcus xinjiangensis. Competent Deinococcus sonorensis were made and a plasmid extract was done on D. sonorensis too. 


Methods

    On Monday, Gram stains are performed on D. xinjiangensis grown on three different plates: one of R2A, and two of TGY. The bacteria grown on TGY are gram-positive, while the stain done from R2A reveals contamination by Staphylococcus. Two TGY plates and two R2A plates are inoculated with D. xinjiangensis. 2 flasks of 150mL of R2A are also inoculated with D. xinjiangensis. Two R2A plates are inoculated with D. caeni. The following day, the plates inoculated with D. xinjiangensis are tested through a gram-stain. All of the results are gram-positive. A 200mL flask of R2A is made and OD values are taken of the D. zonorensis inoculated yesterday. On 4/24/24, three 2mL centrifuge tubes containing R2B and D. sonorensis are centrifuged for one minute at 13,000 rpm. This process is repeated five times before cell pellets are visible in the tubes. The cells are then gram-stained. Tube 1 yields an inconclusive stain, while tubes 2 and 3 yield positive gram-staining bacteria. 200 mL of R2A is heated and poured into eight plates. Three flasks of R2B are inoculated with D. sonorensis and four flasks of R2B are inoculated with D. xinjiangensis. The following day the three R2B flasks of D. xinjiangensis are gram-stained. All three contain spherical, tetrad-forming gram-positive bacteria. One mL is taken from each flask and put into a centrifuge tube. The concentration of these tubes is then normalized to have an OD value of 1. 50uL of solution is taken from each tube and placed on parafilm before being exposed to 50,000 uJ/cm^2 of UV radiation. Separate samples are exposed to 100,000uJ/cm^2 of radiation. 20 uL of the sample is then streaked onto a plate of R2A. 20 uL of the non-irradiated samples is streaked onto a separate plate. Plasmid extractions are then done on D. sonorensis. 600uL of R2B and D. sonorensis are placed into two centrifuge tubes and centrifuged. 100uL of Lysis Buffer is mixed into each tube. 350 uL of neutralization buffer is added and mixed into the tubes. The tubes are then centrifuged. 200uL of Endo-Wash buffer is added into the tubes followed by 400uL of Zyppy Wash Buffer followed by 30 uL of Zyppy Elution Buffer, the tubes are then centrifuged. The following day, the plasmid samples are loaded onto an electrophoresis gel and run on 80 amps. The gel shows that no plasmids were extracted from D. sonorensis. OD values are taken for the two flasks of D. sonorensis and yield OD values of 0.32 and 0.24. After centrifuging the cells and resuspending them in 1mL of solution from each sample's respective flask. OD values are taken of each sample and yield values of 0.68 and 0.45. For the first sample, cells are pelleted, the supernatant is discarded, and the cells are resuspended in 1 mL of the solution. This procedure is repeated for the second sample, except that the procedure is donw twice. OD values are taken again, and each sample yields a value of 1.04 and 1.06. These samples are then centrifuged and the supernatant is discarded. The cells are resuspended in 1290 uL of clean R2B. They are mixed with a solution containing 323 uL of 60% glycerol, 335 uL of PCR water, and 51.6 uL of CaCL2. The samples are mixed and stored at -80 degrees Celsius. The flasks of D. sonorensis are then discarded. 

Results

    D. sonorensis showed no evidence of having a plasmid. 

Discussion

   More time is needed to determine the results of the UV test. Because the dilution factor for the non-irradiated cells was so vastly different from that of the irradiated cells, it is imposible to draw meaningful comparions between the growth levels for them both. 

Comments

Post a Comment

Popular posts from this blog

BioTech Lab Entry 6

  Introduction     This week was spent desiging ways to further test the UV resistance of Deinococcus xinjiangensis .  Methods    Six flasks of TGY, each containing 25uL, are prepared. Five flasks of 20 uL of R2B and one flask of 20 uL of R2A is prepared. A practice gel is done for electrophoresis. The gel contains 30mL of TAE buffer and 0.3g of agarose. Cryovials contain 2uL of the sample, 8uL of PCR water, and 2uL of the loading dye. The gel is run at 100 amps. The next day, 2 flasks of TGY and one flask of R2B are inoculated with D. xinjinangensis . They are put in the skaing incubator at 30.0 degrees Celsius. I then had a discussion with Dr. Tuohy on further avenues of research (see discussion). Gram stains are done on the flasks inoculated yesterday. However, the results are inconclusive. Aland and I design a new and simplified experimental design for the UV tests that can be easily manipulated to test for specific variables. The following day, A...
Read more

GCC BioTech Lab Blog Entry 2

Introduction     This week in the lab was not very busy. We hoped to innoculate plates and perform characterization tests on Deinococcus xinjiangensis , but were unable to do so. Our group also began investigating whether or not certain proteins involved in mechanotaxis and chemotaxis were present in various Deinococcus  species.  Methods       28 plates were prepared. 10 contained TGY, two contained a comibination of starch and TGY, six contained urease, and 10 contained R2A. 500 ml of TGy was heated at 350 C and poured into the TGY plates. Another TGY flask of the same volume was heated at 355 C and poured. 2g/L of strach and TGY was used to make the starch plates, while the solution for the urease test consisted of 50mL of urease in 450mL of H20 and Agar after the latter solution was heated to 350 C. After the plates were poured they were placed in the refrigerator. Next a R2B broth was prepared. 0.378g of R2B powder was dissolved in 120 mL of DI wa...
Read more