Paul.xu at 14:10, 2 January 2013

2013-01-02T14:10:30Z

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== 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) <strike>5 yeast extract</strike> 

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 MgSO''''''4'''

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. 

 

 

 

[[Category:Protocols]] [[Category:Buffer_Recipe]]

AutoInduce Protocol - Revision history

Paul.xu at 14:10, 2 January 2013