This protocol is a larger scale variant of one due to Mike
Scott, Department of Neurology, UCSF.
Streak out the desired strain on an LB plate. Pick colony
and grow in 3mL LB over night. (If you are using SureTM cells, then use
LB+Tetracycline.)
The next day, inoculate 100 ul into 20 ml TYM broth
in a 250 ml flask. Preheat all broth solutions to 37ºC if you want to be done before midnight.
Grow the cells to midlog phase (OD600 between 0.2 - 0.8), pour into
2 L flask containing 100 ml TYM, continue vigorous agitation until cells grow to 0.5 - 0.9 OD, then dilute again to 500
ml in the same vessel.
When cells grow to an OD600 of 0.6 put the flask in ice-water, and swirl
contents gently to assure rapid cooling. When culture is cool, pellet the
cells (spin 2.5 krpm for 15 minutes in the JA-10).
Pour off the supernatant, resuspend the pellet in 100 ml cold TfB
I by gentle shaking on ice. Re-pellet the cells in the same bottle
(spin 2.5 krpm for 8 minutes in the JA-10 rotor).
Pour off supernatant, and resuspend the pellet in 20 ml cold TfB II by gentle shaking on ice.
Dispense in 250 ul aliquots in prechilled microfuge tubes,
freeze in liquid nitrogen, and store at -70oC. For
transformation protocols, click here