"
Team:UNC Chapel Hill/31 July 2009
From 2009.igem.org
Notes
- Acid washed two 2 L beakers.
- Added about 150 mL of LB to each one.
- Marked one to be ccdBr and the other as DH5alpha. Added 5 mL of the appropriate culture to each one.
- Put on the Shaker at 37 degrees. Checked the OD every 20 minutes until the OD was at least 0.4.
- Took the beakers out and put them on ice. Had dH20, 10% glycerol, 50 mL centrifuge tubes, and DYT on ice as well.
- Waited 25 minutes and then transferred the beaker solutions to the appropriate centrifuge tubes.
- 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.
- Centrifuged for 20 minutes at 1000g. Decanted. Added 10 mL of cold dH20 and vortexed to disperse pellet.
- Centrifuged again for 20 minutes at 1000g. Decanted. Added 10 mL of gold 10% glycerol.
-KS&EK
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
