Standard Cell Preparations

  1. Begin 1L prep by growing a 5 ml overnight culture.

    1. Using a sterile, plastic pipette, dispense 5 ml of 2xYT media into a sterile Falcon tube. Flame sterilize media bottle before putting it away.

    2. Add 5 ul of 100mg/ml stock of filter-sterilized ampicilin.

    3. Take a frozen chip from the cell stock you are to use using a sterile toothpick. Always flame sterilize the tweezers you use to handle the sterile toothpick.

    4. Drop the toothpick into the culture media and flame sterilize the Falcon tube again.

    5. Place the overnight cultures into the incubator shaker and incubate 10-16 hrs at 37oC.


  2. Add 1L of sterilized 2xYT media to an autoclaved 2.8L baffled culture flask.

  3. Add 1 ml ampicilin and your overnight culture, flame the flask, cover with aluminum foil and place in a 37oC shaker.

  4. Let bacteria grow 16-24 hrs before harvesting.

  5. Before spinning down the cells, take 1 ml out to check the OD600 against fresh 2xYT media. Record this value.

  6. Take another 250 ul out to check protein expression.

    1. Spin the aliquot at 6000 rpm for 5 min.

    2. Decant supernatant, add 50 ul water and 50 ul 3xSDS sample buffer and resuspend pellet with pipette tip and vortexing.

    3. Boil the samples at 100oC for 20 min before running on SDS-PAGE.


  7. Spin cells at 6000 rpm for 20 min, pour of supernatant and freeze pellet.

  8. Wash flasks and centrifuge tubes using bleach.

  9. The next day, remove the frozen pellets from the flasks and store them in 50 ml conical tubes until a protein prep is performed. Wash and put away the centrifuge flasks.

  10. Two bits of information needed for each culture grown.

    1. OD600 at time of harvesting.

    2. SDS polyacrylamide gel to insure protein expression.