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



Revision as of 20:45, 3 September 2013

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.