Team:UNC Chapel Hill/31 July 2009




  1. Acid washed two 2 L beakers.
  2. Added about 150 mL of LB to each one.
  3. Marked one to be ccdBr and the other as DH5alpha. Added 5 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 0.4.
  5. Took the beakers out and put them on ice. Had dH20, 10% glycerol, 50 mL centrifuge tubes, and DYT on ice as well.
  6. Waited 25 minutes and then transferred the beaker solutions to the appropriate centrifuge tubes.
  7. Centrifuged for 15 minutes at 1000g. Then decanted supernatant with an addition of 50 mL of cold dH20 in each container. Used a 9 mL to break up pellets.
  8. Centrifuged for 20 minutes at 1000g. Decanted. Added 10 mL of cold dH20 and vortexed to disperse pellet.
  9. Centrifuged again for 20 minutes at 1000g. Decanted. Added 10 mL of gold 10% glycerol.


spun down again, resuspended each tube in ~300 uL GYT. Checked OD (.35). Checked for arcing-arced. Spun down again, decanted, resuspended in ~300uL GYT. Distributed into individual 1.5mL epi tubes, flash froze in dry ice/ethanol bath. Placed in -80. -ML