The day before making the cells
1) Streak out a plate of the strain of interest from a -70°C freezer stock and grow overnight at 37°C.
2) Prepare 1000 mL of LB medium in a 2.8 liter fernbach flask. Autoclave this, along with a 4 500 mL centrifuge bottles and 4 35 mL centrifuge tubes.
NOTE: DO NOT AUTOCLAVE THESE TUBES & BOTTLES WITH THE LIDS ON - INSTEAD AUTOCLAVE SEPARATELY EITHER WRAPPED OR COVERED WITH FOIL.
2) Prepare 100 mL of 10% glycerol (must be autoclaved as well)
4) Prepare 6 500 mL bottles of sterile MQ H2O (must be autoclaved as well)
Making the competent cells
1) Prechill the sterile MQ H2O and glycerol on ice.
2) Inoculate a single colony from the overnight plate into the 1000 mL of LB medium.
3) Grow at 37 °C/250 rpm until A600 reaches 0.55.
4) Transfer the cells to the sterile 500 mL centrifuge bottle (250 mL in each bottle). Gently pellet the cells (JA10, 8 krpm, 6 min, 4 °C)
5) Carefully dump off the supernatant; Drain the pellet by standing the bottle upside down on a clean paper towel.
6) Resuspend the pellets in 250 mL of ice-cold MQ H2O. Gently pellet the cells (JA10, 8 krpm, 10 min, 4 °C). Note, the cell pellet is quite soft at this point. Use care in dumping off the supernatant!
7) Carefully dump off the supernatant. Resuspend the pellets in 250 mL of ice-cold MQ H2O.
8) Gently pellet the cells (JA10, 7 krpm, 6 min, 4 °C). Carefully dump off the supernatant.
9) Carefully dump off the supernatant. Resuspend the pellets in 250 mL of ice-cold MQ H2O.
10) Gently pellet the cells (JA10, 7 krpm, 6 min, 4 °C). Carefully dump off the supernatant.
11) Resuspend the pellets in 20 mL of sterile ice-cold 10% glycerol. Transfer to the sterile 50 mL centrifuge tube. Pellet the cells: JA-17, 8 krpm, 10 min, 4 °C.
12) Carefully dump off the supernatant. Resuspend each pellet in 0.5 mL 10% glycerol; Pool the cells into a single centrifuge tube. Transfer 50 mL aliquots to prechilled sterile eppendorf tubes and freeze at -70 °C.
