Sp'23: Week 9 (3/20/23 - 3/24/23): Continuing with Attempted Growth

As spring break has come to an end, it is now time to start working with transformation once more. With there being just two months left of school, I wanted to make sure I was on the right track, so I spent some time planning and brainstorming with Dr. Tuohy about how to organize the rest of the semester. Ideally, one transformation will be run each week, but depending on growth / availability / potential contamination, a number of factors can play into an ideal timeline. 

Along with this, I created a 1.5% agarose gel that was eventually placed into the fridge (this gel will be available to do a gel run after any future extractions and plasmid lysis I do in the future). I typically make 1% gels consisting of the following materials: 

  • 30 mL 1X TAE Buffer 
  • 0.3 g Agarose 
  • [mixed in a 125 mL Erlenmeyer flask]
However, this time I made a 1.5% agarose gel for electrophoresis that was made of the following materials instead (same ingredients, different amount of Agarose): 
  • 30 mL 1X TAE Buffer 
  • 0.45 g Agarose 
  • [mixed in a 125 mL Erlenmeyer flask] 
Agarose is a polysaccharide that, when used to make gels, create small pores through which DNA can pass through when the electric current passes through the gel during a gel electrophoresis run. The meaning of 1% involves the ratio of materials to eachother, with a higher percentage gel being one that has smaller pores as there is a higher concentration of agarose in the gel. These smaller pores make it easier for smaller pieces of DNA to run through and more difficult for larger pieces of DNA to run through, so because of this, it would be easier to visualize any smaller fragments of plasmid that may have been procured with a plasmid extraction done on E. coli with the plasmid pRad1 (which is the plasmid I am using to attempt transformation on Deinococcus aquaticus). 

This week, I also inoculated potentially-transformed Deinococcus aquaticus to new sets of TGY. The following four tubes were created then placed into the 30°C incubator for growth (and names were as follows): 
  • "3.22A1, TGY 3:3:1 +Cm [3 ug/mL] from tube 3.3P1, 3/22/23 M.O."
  • "3.22A2, TGY 3:3:1 +Cm [3 ug/mL] from tube 3.3P2, 3/22/23 M.O." 
    • Both tubes 3.22A1 and 3.22A2 came from tubes 3.3P1 and 3.3P2, both of whose bacteria had been originally inoculated from potentially transformed plates 
  • "3.22B1, TGY 3:3:1 +Cm [3 ug/mL] from tube 3.3B1, 3/22/23 M.O." 
  • "3.22B2, TGY 3:3:1 +Cm [3 ug/mL] from tube 3.3B2, 3/22/23 M.O." 
    • Both tubes 3.22B1 and 3.22B2 came from tubes 3.3B1 and 3.3B2, both of whose bacteria had been originally inoculated from tubes 3.3P1 and 3.3P2 
Now, the growth of these four tubes with potentially-transformed D. aquaticus in TGY was checked two days later, with OD600 readings from the nanodrop as follows: 

These are extremely low growth readings, as the A600 numbers would be expected to be closer to 1 to show decent growth. The growths of the four tubes (3.22A1, 3.22A2, 3.22B1, and 3.22B2 seen in this order) are shown above. Because the growth was extremely low, all four tubes were placed back into the 30°C incubator to see if they will grow more over the weekend.

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