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:
- 64g Na2HPO4-7H2O
- 15g KH2PO4
- 2.5g NaCl
- H2O to final 1L volume and autoclave
To prepare 500mL M9 minimal media;
- 100mL of 5xM9 salts
- 1 mL 1M MgSO4
- 50 uL 1M CaCl2
- 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).
- 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).