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From 2013.igem.org

1. Grow media culture of the cells at 37 degrees
2. Inoculate flask with 500mL of SOB media with 1mL of the media culture
3. Place flask in the 37 degree Celsius shaker.
4. Check the OD reading about every hour
5. Once the OD reaches between 0.3 and 0.4, the rest of the protocol can be followed.
6. Put the flask on ice
7. Split the contents in the flask into 2 250mL centrifuge tubes
8. Centrifuge at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Make sure to adjust the rotor setting.
9. Decant the supernatant and resuspend each pellet in 100 mL of ice cold ddH2O.
10. Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees
11. Decant the supernatant and resuspend each pellet in 50 mL of ice cold ddH2O.
12. Combine resuspensions into 2 centrifuge bottles . Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. At this step,

rinse two 50 mL conical tubes with ddH2O and chill on ice.

1. Decant the supernatant and resuspend each pellet in 20 mL of ice cold 10%

glycerol. Transfer each suspension to a 50 mL conical tube.

1. Harvest the cells by centrifugation at 1000 RCF, 3 Decel, 9 Accel, 4 degrees. Start putting 1.5 mL microfuge tubes on ice if not

already chilled.

1. Carefully aspirate the supernatant with a sterile Pasteur pipette (pellets lose

adherence in 10% glycerol). Resuspend each pellet in 1 mL of ice cold 10% glycerol by gently swirling. The final OD600 of the resuspended cells should be ~ 200-250.

1. Aliquot into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen.

Store frozen cells in the -80°C freezer.