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Preparation of Broth and Plates, etc.

[Use ddH2O 17 m* or better ]

Recipes:

1) LB Broth

Make 16 gm of LB Broth Base (Gibco #M27800C) up to 800 ml in ddH2O.

Swirl to dissolve, then add 110 µl of 10 N NaOH. Autoclave.

2) NZY Broth Combine:

NaCl 4 gm (Fisher #S671-500; from Material Manag.)

MgSO47H2O 1.6 gm (Sigma #M-2773)

Yeast Extract 4 gm (Difco #0127-05-3)

N-Z-Amine 8 gm (Sigma #C-0626)

Make up to 800 ml in ddH2O. Swirl to dissolve, then add 110 µl of 10 N NaOH. Autoclave.

3) Terrific Broth Combine:

Tryptone 9.6 gm (Difco #0123-05-7)

Yeast Extract 19.2 gm (Difco #0127-05-3)

Glycerol 3.2 ml (Sigma #G-5516)

Make up to 720 ml in ddH2O. Swirl to dissolve, then autoclave 20 min.

Add 80 ml of 0.17 M KPO4 (make 1.85 gm KH2PO4 and 10 gm K2HPO4 up to 80 ml in ddH2O; autoclave) and autoclave.

4) LB Plates

Make 25.5 gm of LB Agar (Gibco #M27000C) up to 800 ml in ddH2O.

Swirl, then add 110 µl of 10 N NaOH. Autoclave, then mix well.

Allow to cool to 50°C (check with BioRad temperature indicator strip) then add 800 µl of 50 mg/ml ampicillin for LB-Amp [red/black], or 4 ml of 10 mg/ml kanamycin for LB-Kana [red/red], or 1.92 ml of 5 mg/ml tetracycline for LB-Tetra [black/black].

Mix well by swirling, then immediately pour onto plates by flame. Add just enough to cover bottom. Flame surface to remove air bubbles.

5) NZY Plates Combine:

NaCl 20 gm

MgSO47H2O 8 gm

Yeast Extract 20 gm

N-Z-Amine 40 gm

Bacto Agar 60 gm (Difco #0140-01; from Material Manag)

Make up to 4000 ml in ddH2O. Swirl, then add 900 µl of 10 N NaOH. Autoclave for 75 min. Cool to 50°C. Pour under flame onto 150 mm plates.

6) NZY Top Agarose

Add 1.33 gm agarose (Pharmacia #17 0554-02) to 190 ml NZY broth. Autoclave. Cool to 47°C. Solidified, sterile top agarose can be microwave melted, then cooled to 47°C.

7) Antibiotics

Ampicillin stock, 50 mg/ml in ddH2O, filter sterilize, aliquot and store at -20°C.

Kanamycin stock, 10 mg/ml in ddH2O, filter sterilize, aliquot and store at -20°C.

Tetracycline stock, 5 mg/ml in ethanol (no steril.); store at -20°C.

8) SM Buffer Combine:

NaCl 4.64 gm

MgSO47H2O 1.6 gm

1 M Tris, pH 7.5 40 ml

2% gelatin 4 ml

Make up to 800 ml in ddH2O. Mix, then autoclave 30 min.

Dispense in 50 ml aliquots.

For 2% gelatin (Sigma #G-9391), make 3 gm up to 150 ml in ddH2O.

For 1 M Tris, pH 7.5, make 96.9 gm of Tris base up to 640 ml in ddH2O. Add 52 ml of concentrated HCl, check pH with a small aliquot. Make up to 800 ml.

9) 1 M MgSO4

Make 49.3 gm MgSO47H2O up to 200 ml in ddH2O; autoclave.

10) 20 % Maltose

Make 20 gm up to 100 ml in ddH2O; filter sterilize.


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