- Grow a > 2 mL liquid culture to linear phase (you will need 0.6 mL culture per stock tube).
- Transfer 0.6 mL to a 1.5 mL tube. Spin at top speed for 3 min. at room temp.
- Discard the supernatant (to get rid of antibiotic, which can become toxic to stored cells).
- Resuspend the pellet in 0.6 mL plain LB or SOC medium.
- Transfer the cells to a cryogenic storage tube.
- Add 0.4 mL 50% glycerol to the cells for a final concentration of 20%.
- Store at -80°C.
To recover cells from frozen stocks
- Pre-warm an agar plate with the appropriate selection antibiotic at 37°C.
- Bring with you to the -80°C freezer: an ice bucket, clean micropipette tip, and the agar plate.
- Place the frozen stock tube on ice.
- Open the stock tube, quickly scrape up some of the cells with the pipette tip, and streak the agar plate.
- Put the tube back into -80°C (do not let the stock cells thaw).
- Grow the cells at -37°C.
- Pick from the streak (or a colony) and grow in liquid medium to do a mini prep.