242
ANNETTE DEICHMANN AND KLAUS DEICHMANN
Media 2 x YT medium
HzO
Bacto-tryptone (1.6%)
Bacto-yeast extract (1%)
NaCl (1%)
Adjust pH to 7.3 with 1 M NaOH.
Add H20 to make 1 liter.
Sterilize in the autoclave.
950 ml
16 g
10 g
10 g
For casting 2 x YT plates follow steps 2-7 in Subprotocoll, Materials, without adding antibiotics.
Agar 2 x YT top-agar
To lliter 2 x YT medium add 7 g agar. Sterilize in the autoclave and cool to
48°C if you need the top-agar the same day. Otherwise you can store the
autoclaved top-agar at room temperature. Before use it must be melted
in the autoclave or microwave oven and cooled to 48°C.
Casting plates Casting minimal agar plates
1. Prepare the following salt solution:
Na2HP04 · 2 H 20 (0.7%)
KH2P04 (0.3%)
NaCl (0.05%)
NH4Cl (0.1%)
Add H20 to 950 ml.
Adjust pH to 7.4 with 10 M NaOH.
7g
3g
0.5 g
1 g
2. Add 15 g agar, autoclave and cool to 60°C.
3. Add the following solutions:
20% glucose (sterile-ftltered)
10 ml
10 mM CaClz (autoclaved)
10 ml
1 M MgS04 (autoclaved)
1 ml
Thiamine (100 (g/ml; sterile-filtered)
50 J..Ll
Add sterile H20 to make 1 liter.