AutoInduce Protocol

Protocol 1

1. Autoclaved Medium:

Phosphate Buffer, pH 7.2: 6 g Na₂HPO₄/3g KH₂PO₄

Tryptone 20 g

Yeast Extract 5 g

NaCl 5g

Autoclave

2. 50 x 5052

25% glycerol, 250 g/L
2.5% glucose, 25 g/L
10% alpha-lactose, 100 g/L

Glycerol should weight, and add some water to disolve; then add glucose and lactose (heat to disolve);

autoclave, store at 4 oC

Add 20 ml of 50X 5052 to 1 L Autoclaved Medium, Add antibiotics and 1 drop of antiform

Protocol 2

1.LBY

25 g LB powder (or 1% tryptone 10 g/L)
5 yeast extract

dd water to ~ 927 mL.

Autoclave.

2. 20 x P
1.0 M Na₂HPO₄-7H₂O: 268 g/L
1.0 M NaH₂PO₄-H₂O: 139 g/L

optional:  0.5 M (NH₄)₂SO₄, 66 g/L, for room temperature.

Add in order, water first. At last, adjust the volume to 1L with water.

Autoclave

3. 50 x 5052

25% glycerol, 250 g/L
2.5% glucose, 25 g/L
10% alpha-lactose, 100 g/L

Glycerol should weight, and add some water to disolve; then add glucose and lactose (heat to disolve);

autoclave, store at 4 oC

4. 500 x MgSO4
1 M MgSO₄-7H₂O 24.65 g/100 ml

To make autoinduction medium

927 ml LBY

50 ml 20 x P

20 ml 50 x 5052

2 ml 500 x MgSO₄

1~2 ml 1000X antibiotics (Kan, or Amp or Kan/Chol  or Amp/Chol)

1 tiny drop of antiform (in 70% ethanol)

1. at 9 AM, pick a single colony, grow in 4 ml LB/antibiotics

2. In the afternoon, make 1 to 8 L autoinduction medium.

3. add 0.5 to 4 ml bacteria to autoinduction medium

4. Grow bacteria at 37 oC O/N.

5. Next day 9am ~ 11:00am or 3:00pm: spin down bacteria using 500 ml centrifuge tube, 6000 rpm for 5 to 10 min.

6. wash bacteria with de-ionized water, transfer to 50 ml centrifuge tubes, spin down at 6000 rpm for 5 min.

7. store bacteria at -20 oc or -80 oc.

Another protocol

1% Bacto-tryptone, 0.5% Bacto-yeast extract, 40 mM K2HPO4, 10 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM Mg2SO4, 0.5% glycerol, 0.05% glucose, 0.2% lactose, trace metals

1) Mix together in a 2 Liter Erlenmeyer flask :
898 ml ddH2O
10 g Bacto-tryptone
5 g Bacto-yeast extract
2.68 g NH4Cl
0.71 g Na2SO4
5 g Glycerol
0.5 g Glucose
2 g Lactose

2) Autoclave using a Liquid cycle for 45 minutes.

3) Cool to room temperature, then add:
1 ml 2 M MgSO4 (0.22µm FILTER STERILIZED)
1 ml 1000X Trace Metals Mix (0.22µm FILTER STERILIZED)
40 ml 1 M K2HPO4 (0.22µm FILTER STERILIZED or AUTOCLAVED)
10 ml 1 M KH2PO4 (0.22µm FILTER STERILIZED or AUTOCLAVED)

4) Add the appropriate antibiotic(s) and the media is ready to use.