Preparation of Freezing Medium (8 liters)
1. Clean 2 4000ml plastic beakers with Double Distilled water(DD H2O)
2. Add approximately 3000ml DD H2O to each beaker.
3. Place beakers on stir plates with large stir bars and turn stir plates on.
4. Add 100g Luria-Bertani Broth (LB) powder or granules to each beaker,
5. Add 25.1g of Dibasic Potassium Phosphate (K2HPO4) to each beaker.
6. Add 7.2g of Monobasic Potassium Phosphate (KH2PO4) to each beaker.
7. Add 2g of Sodium Citrate (Na3C6H5O7 ∙ 2 H20) to each beaker.
8. Add 3.6g of Ammonium Sulfate ((NH4)2SO4) to each beaker.
9. Add 176ml Glycerol to each beaker. Because of the high viscosity of Glycerol it is best to add 176ml of Glycerol to a 200ml graduated cylinder and add the contents to the beaker. Then rinse the remaining Glycerol into the beaker by filling the graduated cylinder twice with DD H20, and emptying the contents into the beaker.
10. Bring the total volume in each beaker up to 4000ml by adding DD H20.
11. Stir the beakers again briefly, and divide the contents among 5 clean 2000ml media bottles, adding 1600ml to each.
12. Loosen the caps on each bottle and apply autoclave tape.
13. Label each bottle with media type, the group name and date prepared.
(Example… FM BAC Res. 09-03-02)
14. Autoclave the media on the liquid cycle for 30 minutes.
15. Allow the media to cool to room temperature before use. May store for weeks at RT.
Immediately before use, two additional chemicals must be added.
1. Using a micropipette, add 640 micro liters of Magnesium Sulfate (1M MgSO4 ∙ 7 H2O) to each 1600ml bottle of freezing medium to be used. (final conc is 400uM)
2. Add antibiotic appropriate to the use, often 400 micro liters of Chloramphenicol 50 per 1600ml of medium. Final conc. of Chloramphenicol is 12.5ug/ml.
3. Mix thoroughly by swirling the media inside of the bottle.
Woo et al. Nucleic Acids Res. 22:4922-4931. 1994