Team:Waterloo/Notebook/Recipes

Making LB Media
Materials  500 mL media bottle or rack of test tubes 1 L beaker Magnetic stir bar Tryptone Yeast extract Sodium chloride Granulate agar (only for solid media)

Instructions  1. Fill 1 L beaker with 500mL of deionized water. 2. Add magetic stir bar into beaker and on to magnetic stirrer. Turn to medium speed. 3. Measure and add slowly with a scupula: - 5g tryptone - 2.5g yeast extract - 2.5 g sodium chloride 4. Once dissolved in the deionized water, pour in media bottle or aliquot 5mL in tubes. 5. If making solid media, add 7.5g granulated agar to the media bottle. DO NOT add it to the beaker. 6. Autoclave slightly open bottle/tubes on slow exchange.

Making Auto Induction (AI) Media
Before you start  Certain parts are on variable copy number plasmids. The plasmid is normally low copy number, but when lactose is used to induce, the plasmid becomes high copy number. Auto-Induction media causes this to happen conveniently by providing glucose and lactose. The cells will feed on glucose and replicate to high density. The glucose will then be exhausted and the lactose will induce the plasmid to become high copy number. The lactose does not induce the plasmid before it is at high density thanks to catabolite repression.

Materials  500 mL media bottle 1L beaker Magnetic Stirrer Na2HPO4 KH2PO4 Yeast extract NaCl Glycerol NaOH 10% glucose solution 8% lactose solution

Instructions  1. Fill 1 L beaker with 500 mL of deionized water. 2. Put on magnetic stirrer. 3. Measure and add to beaker: - 3g Na2HPO4 - 1.5g KH2PO4 - 2.5g yeast extract - 2.5g sodium chloride - 3g glycerol 4. Adjust pH to 7.2 with NaOH using a pH-meter. 5. Fill the media bottle. 6. Autoclave with slightly open lid on slow exchange. 7. Filter sterilize 10% glucose solution. 8. Filter sterilize 8% lactose solution.

At the time you are ready to prepare a culture: 1.Add 12.5mL of lactose solution to the autoclaved stock. 2.Add 2.5mL of glucose solution to the autoclaved stock. - If preparing 5 mL culture tubes, use 125µL, and 25µL, respectively.

Making SOB Medium
Used in growing bacteria for preparing chemically competent cells

Ingredients  - 0.5% (w/v) yeast extract - 2% (w/v) tryptone - 10 mM NaCl - 2.5 mM KCl - 20 mM MgSO4

Per liter: - 5 g yeast extract - 20 g tryptone - 0.584 g NaCl - 0.186 g KCl - 2.4 g MgSO4

Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.

SOB medium is also available dry premixed from Difco, 0443-17.

Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.