Haynes:Glycerol Stocks

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Glycerol Stocks

  1. Grow a > 2 mL liquid culture to linear phase (you will need 0.6 mL culture per stock tube).
  2. Transfer 0.6 mL to a 1.5 mL tube. Spin at top speed for 3 min. at room temp.
  3. Discard the supernatant (to get rid of antibiotic, which can become toxic to stored cells).
  4. Resuspend the pellet in 0.6 mL plain LB or SOC medium.
  5. Transfer the cells to a cryogenic storage tube.
  6. Add 0.4 mL 50% glycerol to the cells for a final concentration of 20%.
  7. Store at -80°C.

To recover cells from frozen stocks

  1. Pre-warm an agar plate with the appropriate selection antibiotic at 37°C.
  2. Bring with you to the -80°C freezer: an ice bucket, clean micropipette tip, and the agar plate.
  3. Place the frozen stock tube on ice.
  4. Open the stock tube, quickly scrape up some of the cells with the pipette tip, and streak the agar plate.
  5. Put the tube back into -80°C (do not let the stock cells thaw).
  6. Grow the cells at -37°C.
  7. Pick from the streak (or a colony) and grow in liquid medium to do a mini prep.