Week of 4/22/24-4/26/24 Remaking Media, Urease Plates, and Competent Cells

 Upon returning to lab, we observed that our previous media containing R2B had reached a timeout due to not being autoclaved properly. In the future, more emphasis will be accrued to ensuring media and respecting specimens are to be autoclaved. Therefore, we had to once again remake R2B media and TGY media. We curated three flasks of R2B with Deinococcus Sonorensis in order to perform plasmid extraction, if there was a plasmid to extract in the first place. We did this via the zippy kit. We also poured urease plates, and inoculated four flasks of R2B with Xinjiangensis to continue forward with our UV testing. Sonorensis was observed to have formed white plaque within R2B, possibly due to the TGY component therein. We then took 1ml each in eight epindorph tubes. Centrifuged that for 1 minute at 13,000 rpm. Thereafter discarding the super natent, ensuring OD values of 1, and repeating the process of centrifuging if needed to enlarge the bacterial pellet. We made it a point to inoculate different bacterial colonies within Sonorensis from the R2B broth. Gram stains were performed on Xinjiangensis meanwhile, in order to ensure contamination had not been an issue throughout our redoing the process for UV testing. Upon observing initial OD values of the latter, #1 had garnered 0.67 OD, thus too low so we had added more broth and homogenized that prior to centrifuging. #2 had an initial OD value of 1.00. #3 had an OD value of 0.67, so once again we added more broth that consisted of Sonorensis, homogenized, then centrifuged that. #4 had an OD value of 1.30, so we added 200ml of clean broth in order to bring that value down. To which came down to an OD value of 1.20, and we repeated the process of adding more clean broth to bring that value down to an acceptable OD value (0.95-1.05). At the end of the weak we researched about competent cells and how to curate such. Competent cells have a permeable cell wall so it is easier to infiltrate such in order to transform cells. To make competent cells we mixed 60% glycerol calcium chloride, R2B, and 2ml of PCR water solution, 2ml of loading dye, 8ml of sample which is what the plasmid consisted of. 

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