Editing Minimal Medium (section)

== Recipe 3 for 15N labeling<br>  ==

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Expression Protocol in M9 Minimal Media via T7 Promoter 

<br>The following protocol has been used successfully to 15N or 13C/15N label proteins using&nbsp; pET/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. If a labeled nitrogen source,&nbsp; 5x salts should be prepared without NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: 

64g Na2HPO4-7H2O<br> 15g KH2PO4<br> 2.5g NaCl<br> H2O to final 1L volume and autoclave 

To prepare 500mL M9 minimal media; 

100mL of 5xM9 salts<br> 1 mL 1M MgSO4<br> 50 uL 1M CaCl2<br> 5 mL 100x Basal Medium Eagle Vitamin Solution (Gibco)<br> 2.5 mL filter sterilized NH4Cl (0.2 g/mL) or 0.5g dry<br> 10 mL 20% d-glucose or 2g dry<br> glass distilled &amp; autoclaved H2O to final volume of 500mL 

pH solution to 7.3 and filter sterilize (0.2u filter). 

Introduce media to a pre-autoclaved, wide-bottom (baffled) 2L flask and add ampicillin to a final concentration of 70-100 ug/ml. 

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)<br> 

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