Freezing Bacterial Stocks

  1. Spin an overnight saturated culture at 2500rpm for 12 min at 4°C. Discard the superna tant.

  2. For a 5 ml culture, add 250 ul of sterile 30% glycerol and 250 ul of L broth. (Add antibiotic, if necessary. Antibiotic stocks are made as a 1000x stock; 1ml of L broth and 1 ul antibiotic. See ampicillin , chloramphenicol , and tetracycline for preparation of antibitic stocks).

  3. Freeze in boxes in the -70°C freezer.

  4. Enter description of bacteria/plasmid in freezer log book.


RECIPES

30% Glycerol
30 ml 100% glycerol
70 ml water
Autoclave.
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
Ampicillin Stock Solution, 100mg/ml
1.00 g sodi um ampicillin (stored in 4° dessicator; Bristol Polycillin-N, 1g/vial; or Sigma
10 ml of autoclaved water
Store at - 30°C in sterile Falcon tube
Chloramphenicol Stock Solution (100 mg/ml) WARNING: Carcinogen.
1 g chloramphenicol dissolved in absolute ethanol.
QS to 10 ml with ethanol. Store at -20°C.
Tetracycline Stock Solution (20mg/ml; 1000X stock)
0.2 g tet racycline
Dissolve in 10 ml of methanol and store in -30°C freezer in a sterile tube.
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Freezing Bacterial Stocks. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/bactfrez.html). 1997. Oklahoma City. Revision Date: October 2, 1997."