Sp'23: Week 2 (1/23/23 - 1/27/23): Bootcamp + Preparation

This week (Week 2) officially begin my journey intro transformation of Deinococcus radiodurans once more. It began with tossing out all of my old plates and broths (both cultured and uncultured), as they were over a month old, and this semester will be a new start with all of the material. 

Now, because I am going to be attempting transformation, I first need to make sure I have plasmid to work with. This semester, I will solely be working with the smaller plasmid, pRad1, and will be attempting to use a different extraction kit altogether (which has been said among our lab folk to have a higher yield of plasmid, and which was actually the extraction kit used to pull the plasmid that originally helped our lab mentor Jonathan transform D.rad).

I started out by creating the following: 

  • 100 mL LB + Agar broth (this will be used to create 4 LB Agar plates with an [Amp] of 50 ug/mL, onto which E. coli with pRad1 from freezeback will first be inoculated) 
  • 300 mL LB broth (will be mostly be used for inoculation) 
  • 200 mL 2xLB broth (this will be used to re-inoculate the E. coli with pRad1 from the LB Agar plates, and it having double the concentration of ingredients will ideally help yield a higher growth of D. rad) 
From the 100 mL LB + Agar broth, the antibiotic Ampicillin was added to it to create E. coli's mic concentration of 50 ug / mL. In the end, this helped to create 4 different LB + Agar plates with [Amp] 50 ug/mL. 


After this, a few more things occurred. I inoculated 2 of the LB Agar +Amp plates with E. coli that has the pRadZ1 plasmid (taken from my own freezeback stash) under the hood (using aseptic technique), after which I realized that I had inoculated E. coli with the wrong plasmid (I actually need to do extraction of the pRad1 plasmid). Because of this, I needed to redo inoculation onto the other two LB Agar + Amp plates using Jonathan's E. coli with pRad1 by using Jonathan's own freezeback archive. 

All of the preparation leading up to the end of this week was quite a bit of work (as there was planning involved on the back-end, too). But, I can now say that I have officially inoculated E. coli with pRad1 onto 2 plates (and the other 2 plates with E. coli + pRadZ1 will potentially be used to harvest), leading to four inoculated plates that are ready to go for next week!


Coming up (with help from my lab mentor): Inoculation of E. coli w/pRad1 into two sets of 10mL 2xLB, followed by a nanodrop reading on the next day, with a gram stain and extraction the day after (as well as inoculation of the E. coli with pRad1 from the 2xLB broth into regular LB broth). 

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