- TBD mL of sterile 10% glycerol
- 18 Jan 2024: I made this by weighing out 15 g ACS-grade anhydrous glycerol into a clean reagent bottle that was rinsed twice with tap water and once with distilled water. The label on the bottle of glycerol says the contents are USP, ACS, and Ultra Pure grade, and has been endotoxin tested and is ATP-free.
- TBD mL of sterile LB culture media ("lysogeny broth" with Miller or Lennox modification)
- x
- x
- Grow an overnight culture of E. coli in 150 mL LB in a flask in a shaker incubator: 37°C, 250 rpm.
- Consider picking a colony from a plate of E. coli to start this culture. 18 Jan 2024: I started with 1.5 mL of a liquid culture I had in the fridge.
- In the morning, transfer 25 mL of the overnight culture to a sterile flask containing 125 mL sterile LB.
- Consider pre-warming the sterile flask of LB to 37°C before this step.
- Let grow for 3-4 hours.
- Protocols I see online recommend growing to an optical density (OD) of as close to 0.4 as possible.
- Get two sterile 50 mL conical tubes (also known as “Falcon tubes").
- Incubate the two 50 mL tubes in a beaker of ice-water for 1 hour. From this point onward, keep the culture as close to 0°C as possible without freezing them solid.
- [TBD: Transcribe further steps from my paper notes]
- xx
- Thaw one of the tubes containing a 150 µL aliquot of electrocompetent cells.
- Add 1 µL of the desired plasmid DNA solution into the cells.
- Flick the tube five times to mix.
- Pipette the cells into an electroporation cuvette with a 2 mm electrode gap.
- I used sterile technique here, but some biologists more experienced than me are completely OK not needing sterility for this protocol because bacteria grow so fast that they outcompete most contaminants, which tend to be molds/fungi from spores in the air, and also because we'll be using antibiotic selection later which should kill any environmental bacteria that grow.
- Set the BioRad Pulse Controller to 200 Ω, and the BioRad Gene Pulser to 200 µF and 2.00 kV (i.e. 2000 V).
- Insert cuvette with the electrocompetent cell/DNA mixture into the cuvette holding fixture connected to the Gene Pulser.
- Press and hold BOTH “pulse” buttons until the machine beeps.
- Immediately pipette the cell/DNA mixture into a sterile culture tube containing 5 mL Terrific Broth (preferred), SOC (preferred), LB (adequate).
- Incubate the tube at 37°C for 1 hour.
- During this time, the cells will grow. Those who took up the plasmid DNA will have a chance to begin making the antibiotic resistance factor coded therein, allowing them to survive the subsequent steps of the protocol.
- 21 Jan 2024: I incubated for 4.5 hours because I had another errand to do in between.
- Streak the cells onto agar plates made with the antibiotic for which the DNA codes resistance.
- Incubate plates at 37°C for at least 3 days, checking for growth each day.
- As soon a growth is seen, it is recommended to refrigerate the plates at 4°C to prevent overgrowth.