Preparation of Broth and Plates, etc.

 

[Use ddH2O 17 mΩ or better from Pfister or White/DeSimone labs]

 

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.