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[[Category:Protocols]]
[[Category:Protein expression]]
[[Category:Protein expression]]
[[category:Cell_Culturing]]
===1. General Protocols For Minimal Media Protein Expression===


1. General Protocols For Minimal Media Protein Expression
Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH<sub>4</sub>Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source for 1L 5xM9 salts:


Expression Protocol in M9 Minimal Media via T7 Promoter:The following protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts:
64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O


64g Na2HPO4-7H2O
15 g KH<sub>2</sub>PO<sub>4</sub>
15g KH2PO4
2.5g NaCl


H2O to final 1L volume and autoclave
2.5 g NaCl
To prepare 500mL M9 minimal media:
 
H<sub>2</sub>O to final 1L volume and autoclave
To prepare 500 mL M9 minimal media:
 
100mL of 5xM9 salts:
 
1 mL 1 M MgSO<sub>4</sub>
 
50 uL 1 M CaCl<sub>2</sub>


100mL of 5xM9 salts
1 mL 1M MgSO4
50 uL 1M CaCl2
5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)
2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5g dry
10 mL 20% d-glucose or 2g dry


Glass distilled & autoclaved H2O to final volume of 500mL pH solution to 7.3 and filter sterilize (0.2u filter).
2.5 mL filter sterilized NH<sub>4</sub>Cl (0.2 g/mL) or 0.5 g dry 10 mL 20% d-glucose or 2 g dry
 
Glass distilled & autoclaved H<sub>2</sub>O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).


Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml (or any antibiotics used to select the strains).
Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/mL (or any antibiotics used to select the strains).


Grow 5mL overnight culture in same media to inoculate 500mL M9.
Grow 5mL overnight culture in same media to inoculate 500mL M9.
Shake culture at 37oC until an OD600 of 07+/-0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).
Shake culture at 37 <sup>o</sup>C until an OD<sub>600</sub> of 0.7
± 0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).
 


===2. General Protocols For LB Media===


2. General Protocols For LS Media
For 1 L media preparation


For 1L media preparation,
10 g Bacto-tryptone
10g Bacto-tryptone
 
5g yeast extract
5 g yeast extract
10g NaCl  
 
10 g NaCl  


Sterilized by autoclave
Sterilized by autoclave
Add any antibiotics used to select the strains properly after the medium temperature is blew 37 <sup>o</sup>C.
===3. General Protocols For High-Cell-Density Media===
For 1 L media preparation
50 mM Na<sub>2</sub>HPO<sub>4</sub>.7H<sub>2</sub>O
25 mM KH<sub>2</sub>PO<sub>4</sub> (pH 8.0-8.2)
10 mM NaCl
5 mM MgSO<sub>4</sub>
0.2 mM CaCl<sub>2</sub>
0.1% NH<sub>4</sub>Cl or <sup>15</sup>NH<sub>4</sub>Cl
1.0% Glucose or <sup>13</sup>C-Glucose
To prepare the media, glucose and metal solution are filtered to sterilize. The salt solution is autoclaved and the cooled solution is added by metal solution and glucose.

Latest revision as of 20:34, 21 July 2022

1. General Protocols For Minimal Media Protein Expression

Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source for 1L 5xM9 salts:

64 g Na2HPO4-7H2O

15 g KH2PO4

2.5 g NaCl

H2O to final 1L volume and autoclave To prepare 500 mL M9 minimal media:

100mL of 5xM9 salts:

1 mL 1 M MgSO4

50 uL 1 M CaCl2

5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)

2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5 g dry 10 mL 20% d-glucose or 2 g dry

Glass distilled & autoclaved H2O to final volume of 500 mL pH solution to 7.3 and filter sterilize (0.2 um filter).

Introduce media to a pre-autoclaved, wide-bottom (baffled) 2 L flask and add ampicillin to a final concentration of 70-100 ug/mL (or any antibiotics used to select the strains).

Grow 5mL overnight culture in same media to inoculate 500mL M9. Shake culture at 37 oC until an OD600 of 0.7 ± 0.2 then induce protein expression with the addition of IPTG (0.01-0.1 mM final concentration).


2. General Protocols For LB Media

For 1 L media preparation

10 g Bacto-tryptone

5 g yeast extract

10 g NaCl

Sterilized by autoclave

Add any antibiotics used to select the strains properly after the medium temperature is blew 37 oC.

3. General Protocols For High-Cell-Density Media

For 1 L media preparation

50 mM Na2HPO4.7H2O

25 mM KH2PO4 (pH 8.0-8.2)

10 mM NaCl

5 mM MgSO4

0.2 mM CaCl2

0.1% NH4Cl or 15NH4Cl

1.0% Glucose or 13C-Glucose

To prepare the media, glucose and metal solution are filtered to sterilize. The salt solution is autoclaved and the cooled solution is added by metal solution and glucose.