Media preparation: Difference between revisions
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1. General Protocols For Minimal Media Protein Expression | 1. General Protocols For Minimal Media Protein Expression | ||
Expression Protocol in M9 Minimal Media via T7 Promoter:The following protocol has been used successfully to | |||
Expression Protocol in M9 Minimal Media via T7 Promoter: The following protocol has been used successfully to <sup>15</sup>N or <sup>13</sup>C/<sup>15</sup>N 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 | 64 g Na<sub>2</sub>HPO<sub>4</sub>-7H<sub>2</sub>O | ||
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2.5 g NaCl | 2.5 g NaCl | ||
H<sub>2</sub>O to final 1L volume and autoclave | |||
To prepare | To prepare 500 mL M9 minimal media: | ||
100mL of 5xM9 salts | 100mL of 5xM9 salts | ||
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50 uL 1 M CaCl<sub>2</sub> | 50 uL 1 M CaCl<sub>2</sub> | ||
5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco) | 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco) | ||
2.5 mL filter sterilized | 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 | 10 mL 20% d-glucose or 2 g dry | ||
Glass distilled & autoclaved | 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) 2 L 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 37 <sup>o</sup>C until an OD<sub>600</sub> of | 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). | |||
Revision as of 04:03, 23 December 2012
1. General Protocols For Minimal Media Protein Expression
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 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 LS Media
For 1 L media preparation, 10 g Bacto-tryptone 5 g yeast extract 10 g NaCl
Sterilized by autoclave