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Competent cells preparation
Protocol: Competent Cells
- 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
- Inoculate 10ml LB medium with desired strain (50-200 µl) and incubate overnight at 37°C, 200-300rpm
- 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).
- Pellet bacteria at 4°C, 4000rpm, 10min in 250ml bottles.
Everything is done on ice from this point
- Discard the supernatant and resuspend the pellet with 300ml (total) of Solution A. Incubate on ice for 20min.
- Pellet bacteria again (see step 3) and resuspend the pellet in 60ml of Solution A + 15% Glycerol.
- 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).
Only using half the amount of cells from the original protocol, only using half the solution amount.
Step 2: Optical density measured at 460.
Step 4: Not enough sol. A + glycerol for the second tube. Only ~27ml instead of 30ml.
