Preparation of Broth and Plates, etc.
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.
7) 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.
8) 1 M MgSO4
Make 49.3 gm MgSO47H2O up to 200 ml in ddH2O; autoclave.
9) 20 Maltose
Make 20 gm up to 100 ml in ddH2O; filter sterilize.
