Solutions needed

• 1 liter LB media + 15mM MgCl2: 1 liter LB + 15ml 1M MgCl2 (autoclave together)
• MES: (50mM 2-N-Morpholinoethanesulfonic acid), 5.33g in 500ml ddH20, adjust pH to 6.3 with 5M KOH, aliquote and store at -20°C for many months
• Solution A*: (10mM MnCl2 4H20 0.99g, 50mM CaCl2 3.7g, 10mM MES (pH 6.3) 100ml, q.s. ddH2O ~400ml -> 500ml in total)
• Solution A* + Glycerol: 85ml Solution A*, 15ml Glycerol

* must be made fresh and sterilized by 0.2um filtration

Protocol

1. Inoculate 10ml LB medium with desired strain (50-200 µl) and incubate overnight at 37°C, 200-300rpm
2. Add the 10ml of culture to 1 liter of LB + 15mM MgCl2. Incubate in the shaker 37°C until you can read O.D. (600 lambda) of 0.4-0.6. This step takes about 3 hours, if O.D. > 0.6 start over. At this point cool down Solution A & Solution A + 15% Glycerol at 4°C & chill pipette tips (place on ice).
3. Pellet bacteria at 4°C, 4000rpm, 10min in 250ml bottles.

Everything is done on ice from this point

4. Discard the supernatant and resuspend the pellet with 300ml (total) of Solution A. Incubate on ice for 20min.
5. Pellet bacteria again (see step 3) and resuspend the pellet in 60ml of Solution A + 15% Glycerol.
6. Aliquote the suspension in eppendorf tubes (200 ul/tube) and store at -70°C to -80°C (use cold sterile tubes and immediately put the tubes on dry ice).