11 November 2021, Frank Lee
I started with six 1 mL liquid cultures of agar art plasmid-transformed E. coli DH5α in tubes.
- I poured the contents of each tube into biohazard waste, leaving only the drop or so of culture that remains in the tube due to surface tension.
- I added 1 mL of fresh LB and an equal volume of 80% glycerol to each tube. The glycerol formed a visible thick transparent layer on top of the liquid in each tube. The glycerol does not seem to mix spontaneously with the culture solution.
- I closed each tube tightly.
- I vortexed each tube for 5 seconds. This seemed to mix the culture and glycerol well and make a homogeneous solution.
- I added three microliters of chloramphenicol to each tube, pipetting the droplet of chloramphenicol onto the inside wall of each tube about a centimeter below the top rim.
- I vortexed each tube to mix, ensuring mixing by adjusting vortex speed while watching the liquid in each tube swirl up the inside of the tube wall to engulf the drop of chloramphenicol I put there.
- I incubated the tubes in the shaker at 240 rpm and 37⁰C or 2 hours. The shaker stopped shaking them automatically after 2 hours but I had an errand and only returned to the bench to remove them from 37⁰C after a total of 8 hours.
- I immediately put the tubes into –20⁰C freezer intending indefinite storage.