1. Bacterial Cultures

1.1. Growth media

Bacterial strains are cultured in either Luria-Bertani (LB) broth or 2xTY media

  1. LB broth

  2. is made as follows:

    10 g Bacto-tryptone
    5 g Yeast extract
    10 g NaCl made to a final volume of 1 L using double-distilled H2O.

    This solution is aliquoted into 10x 100ml, then autoclaved at 120 °C at 15 p.s.i. for 20 min. After cooling, filter-sterilised antibiotics are added as appropriate.

  3. 2xTY media

  4. is made as above but contains (per litre of media):

    16 g Bacto-tryptone
    10 g Yeast extract
    5 g NaCl

    The solution is again aliquoted into 10x 100ml before sterilisation.

  5. M9 Minimal media


  6. 5x M9 salts (to a final volume of 1 litre)

    Divide the 5x M9 salts into 200ml aliquots and sterilise (by autoclave).

    To 750ml of sterile deionised water add the following sterile supplement solutions

    Make volume up to 1litre with sterile deionised water.

     

    1.2. Agar plates

    1.2.1. LB or 2xTY plates

    1.5 g of bacto-agar is added per 100ml of nutrient media prior to sterilisation (by autoclave). Antibiotics are added, as appropriate, to the cooling agar solution and mixed thoroughly. 15-20 ml of the molten agar mixture is added per 100 mm diameter petri dish and allowed to set at room temperature before storage. Excess moisture is removed by allowing the plates to dry near the Bunsen flame.

    1.2.2. Minimal plates

    Make up 3% water agar by dissolving 3g bacto-agar in deionised water and then sterilising (by autoclave).

    Whilst water agar is still warm and molten, warm the 1x M9 salts (plus supplements, section 1.1.3.) to approx. 50° C, then add the two together. Add the antibiotic selection and pour the plates.

    1.3. Antibiotic selection

    The following antibiotics can be used:

    Antibiotic

    Stock solution

    Final concentration used

    Ampicillin

    100 mg/ml in H2O

    100 mg/ml

    Carbenicillin

    100 mg/ml in H2O

    100 mg/ml

    Chloramphenicol

    25 mg/ml in 100% ethanol

    25 mg/ml

    Stock solutions of antibiotics are made in MQ water and then filter sterilised. The typical volume of stock solution prepared is 10ml, which is then aliquoted for storage at -20° C.

    1.4. Bacterial cultures

    1.4.1. Glycerol stocks

    Bacterial glycerol stocks are prepared from 10ml overnight liquid cultures grown with antibiotic selection as required. 0.5ml liquid culture is added to 0.5ml 30% (v/v) sterile glycerol in 1.5ml screw-capped vial or eppendorf tubes. The tube is inverted to mix the cells and glycerol solution, then stored at -80°C.

    1.4.2. Liquid cultures

    Bacterial cultures are inoculated using a wire loop, either from freshly transformed bacteria grown on agar plates or from existing bacterial glycerol stocks. Liquid cultures of less than 100 ml are inoculated directly whereas for larger scale cultures (~1 L), overnight starter cultures of 1-10 ml are used for inoculation. These cultures are incubated at 37°C in an orbital incubator with shaking at approximately 200 revolutions per minute (rpm).

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    Pages created by Dr Sue Jones 30.3.01