S-STEM Scholar Carly Ziegenhagen's Blog

Introduction This week, the results for the UV test done on D. xinjiangensis were observed and a plasmid extraction was performed on D. sonorensis. Procedure The plasmid extraction was performed as follows:  -Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube  -Centrifuge the tube for 30 seconds at maximum speed and discard the supernatant -Add 600 μl of TE or water to the bacterial cell pellet and completely resuspend it -Add 100 µl of 7X Lysis Buffer to the tube and mix by inverting 4-6 times and proceed to the next step within two minutes -Add 350 µl of cold Neutralization Buffer to the sample and mix thoroughly until the sample turns yellow -Centrifuge the tube for 2-4 minutes at 11,000 – 16,000 x g -Transfer the supernatant (~900 µl) to the Zymo-Spin IIN column without disturbing the cell debris pellet -Place the column in a Collection Tube and centrifuge for 15 seconds -Discard the flow-through and place the column back into the collectio...

Introduction This week we reran our UV tests on D. xinjiangensis using R2B media only to grow the bacteria. The purpose of this is to simplify our prior experiment and reduce the amount of variables. We also ran a practice gel electrophoresis. Procedure The procedure for the UV test is as follows: Two R2B flasks were inoculated with D. xinjiangensis  In the following days, bacteria samples were concentrated to an OD 600 value of 1 For each tube sample, a small amount was aseptically transferred onto pieces of parafilm. For each tube, several different samples were placed on parafilm to be exposed to different concentrations of ultraviolet radiation (30000µJ/cm 2 and 100000µJ/cm 2 ) Cell samples were subjected to ultraviolet exposure via the UV Crosslinker Afterwards, the parafilm was aseptically scraped and the potential bacteria inoculated into recovery broth, then incubated After 2 days, the broths were inoculated onto properly labeled R2A plates Results Results are to be seen ...

  Introduction  This week, we retested Deinococcus xinjiangensis for the oxidase and indole tests to confirm our prior results. We also performed UV tests for D. xinjiangensis. In addition to these tests, we also began working on our poster for the ANAS science conference. Procedure Two R2B flasks and 1 TGY flask was inoculated with D. xinjiangensis  The next day, bacteria samples were concentrated to an OD 600 value of 1 For each tube sample, a small amount was aseptically transferred onto pieces of parafilm. For each tube,  several different samples were placed on parafilm to be exposed to different concentrations of ultraviolet radiation (30000µJ/cm 2 and 100000µJ/cm 2 ) Cell samples were subjected to ultraviolet exposure via the UV Crosslinker Afterwards, the parafilm was aseptically scraped and the potential bacteria inoculated into recovery broth, then incubated After 2 days, the broths were inoculated onto properly labeled plates Results The oxidase and...

Introduction Due to the Gibson assembly falling through, we have decided instead to make our project on the characterization of Deinococcus xinjiangensis through biochemical testing. The following tests will be done: Urease test: tests for presence of enzyme urease through observing for the breakdown of urea into carbon dioxide and ammonia byproducts. Indole test: tests for the presence of enzyme tryptophanase through observing for the breakdown of tryptophan into indole and pyruvate byproducts. Starch test: tests for the presence of enzyme amylase through observing for the breakdown of starch into glucose and maltose byproducts. Catalase test: Tests for the presence of enzyme catalase through observing for the breakdown of hydrogen peroxide into water and oxygen gas. Oxidase test: tests for the presence of enzyme cytochrome oxidase through observing for the breakdown of oxidase solution into water and hydrogen peroxide byproducts. Hydrogen sulfide test: tests for a bacteria’s ability...

Introduction This week, we ran a gel electrophoresis on our amplified DNA samples of D. aquaticus from the PCR reaction ran the prior week. The fragments we used were intended to be 7kb long, so the expected result from the gel electrophoresis was two bands measured at the 7kb mark of the ladder. This result would indicate that we were able to successfully isolate the 7kb plasmid. Results The results from the gel electrophoresis showed areas where the dye was smeared across the gel, and no visible bands could be found at the 7kb mark. Bands were visible at two other spots, much lower than 7kb. Discussion These results indicate that the DNA sample isolated is much shorter in base pairs than we intended. The use of gibson assembly, a method allowing for the joining of several DNA fragments into one, will be used the following week in attempts to achieve 7kb plasmid isolation without having to restart the process from inoculation onward.

 Introduction This week, we performed a successful inoculation of D. aquaticus from plate to broth. The broth was used later in the week for DNA extraction using the Zyppy Plasmid Miniprep Kit. Procedure The following steps were followed to inoculate D. aquaticus from plate to broth: - A flask of appropriate size should already have media in it and be labeled with initials, date, bacteria name, and type of media. The flask should have been autoclaved and made ready for inoculation. - Gather a bunsen burner, an inoculation loop, and a plate. - Heat the inoculation loop over the bunsen burner flame to sterilize the loop, and let it cool for about 20 seconds. - Open the flask and have the lid in between your fingers underneath the loop. - Swipe the lip of the flask over the bunsen burner flame to sterilize. - Open the plate just enough to collect one colony with the inoculation loop. Close the plate. - Place the loop with the colony into the flask and sweep it around to spread the bac...