Preparation of Broth and Plates

 

Preparation of Broth and Plates, etc.

<center> <p> <font>[Use ddH2O 17 undefined or better ]</font></p> </center>

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