Altman:Protocols/MB Protocols/Glycerol stock

Starting a bacterial culture from a gycerol stock

• Prepare TB (terrific broth) beforehand. TB can be used for a few weeks, and then must be discarded.

1. Prepare a glass test tube with 5 mL of TB+antibiotic

2. Scrape a chunk of the glycerol stock with a sterile loop, and place in the test tube

3. Shake or spin at 37C for no more than 18 hours

• Tubes should be cloudy with cells. Be sure to do a control, which should appear clear at the end of 18 hours

4. Move the 5 mL of culture to a 15 mL conical and spin in a centrifuge at 4000 rpm for 15 minutes

5. Remove the supernatant

6. If you are not going to do the Miniprep that day, store the cell pellet at -20C