Team:UNC Chapel Hill/7 August 2009

1. Acid washed one 1 L beakers. 2. Added about 500 mL of LB to beaker. 3. Added 25 mL of the appropriate culture to each one. 4. Put on the Shaker at 37 degrees. Checked the OD every 20 minutes until the OD was at least 0.5. 5. Took the beaker out and put it on ice. Had dH20, 10% glycerol and 50 mL centrifuge tubes on ice as well. 6. Waited 30 minutes and then transferred the beaker solution to two centrifuge tubes. 7. Centrifuged for 15 minutes at 6000 rpm. Then decanted supernatant and added 500 mL of cold dH20 in each container. 8. Centrifuged for 15 minutes at 6000 rpm. Decanted, then added 250 mL of cold dH20 and vortexed to disperse pellet. 9. Centrifuged again for 15 minutes at 6000 rpm. Decanted, then added 10 mL of cold 10% glycerol. 10. Centrifuged again for 15 minutes at 6000 rpm. Decanted, then added 2.5 mL of cold 10% glycerol. 11. Checked for arcing, which there was none. Got a time constant of 4.3 ms 12. Distributed into individual 1.5mL epi tubes, flash froze in dry ice/ethanol bath. Placed in -80.

-EK