Sp'23: Week 11 (4/3/23 - 4/7/23): Plate Prep and Inoculation for Future Transformations

After last week's unsuccessful attempts at plasmid extractions, it was back to the drawing board (aka time for fresh inoculations). First, I decided that it would be helpful to have my own fresh batch of Deinococcus aquaticus to pull from, so I took from Jonathan's plate (plate "2.10JH") of D. aquaticus and inoculated onto one of my own plates with TGY + Agar (plate "3.24C6" that had been made less than two weeks ago). I also inoculated from is "2.10JH" plate into four different sets of 4mL TGY 3:3:1 polystyrene tubes (labeled "4.5A1," "4.5A2," "4.5A3," and "4.5A4" respectively). The plate and all four tubes were then placed into the 28 degree Celsius incubator to allow them time to grow: 


I also went ahead and made 6 TGY 3:3:1 Agar plates (named "4.5B1" - "4.5B6") with Chloramphenicol (+Cm) [3 ug/mL], which is assumed for now to be the mic concentration of the antibiotic Chloramphenicol for Deinococcus aquaticus, which in turn is based off of the fact that the mic concentration of Chloramphenicol for Deinococcus radiodurans is 3 ug/mL. These plates were made with the knowledge that they would be used for transformation of D. aquaticus with the pRad1 plasmid in the [hopefully] near future: 

With this, I decided to try to grow fresh E. coli with pRad1 by inoculating from my LB + Amp [50 ug/mL] plate of E. coli with pRad1 into four different LB + Amp [50 ug/mL] tubes, which each held a total volume of 4 mL of LB. These tubes were labeled "4.5C1," "4.5C2," "4.5C3," and "4.5C4" and were placed into the 37 degree Celsius incubator for growth: 

Two days later, I ran plasmid extractions using the the full amount of each of the four tubes ("4.5C1" - "4.5C4") to obtain pRad1 to use for transformation. I did not have time to take dsDNA readings with the nanodrop, so this will be done next week (but the four resulting plasmid extractions in their respective microcentrifuge tubes are seen below): 

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