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Preparation of competent E. coli cells N15 and C13 labelling of proteins in E. coli


Materials

Medium A (per liter)

100 ml M9 medium (10x)
10 ml trace elements solution (100x)
20 ml 10% (w/v) C13-glucose
1 ml 1 M MgSO4
0.3 ml 1 M CaCl2
1 ml Biotin (1 mg/ml)
1 ml Thiamin (1 mg/ml)

antibiotic(s)


M9 medium (10x), (per liter)

60 g
Na2HPO4
30 g
KH2PO4
5 g
NaCl
5 g
N15H4Cl


Trace elements solution (100x), (per liter)

5 g
EDTA
0.83 g
FeCl3 x 6 H2O
84 mg
ZnCl2
13 mg
CuCl2 x 2 H2O
10 mg
CoCl2 x 6 H2O
10 mg
H3BO3
1.6 mg
MnCl2 x 6 H2O

First dissolve 5 g EDTA in 800 ml water and adjust the pH to 7.5. Then add the other components and adjust the volume to 1 L. Sterilize the solution over a 0.22 µm filter.


Stock solutions

1 M CaCl2 (sterilized)
1 M MgSO4 (sterilized)
10% (w/v) C13-glucose (sterilized)
1 mg/ml Biotin (filter sterilized)
1 mg/ml Thiamin (filter sterilized)
200 mM IPTG (isopropyl-b-D-thiogalactopyranoside) (filter sterilized)



Procedure

The growth rate of the culture and the optimal induction conditions can vary significantly depending on the vector, the construct and the solubility of the recombinant protein.

1. Transform the vector to the appropriate E. coli strain [e.g. BL21 (DE3) for vectors containing a T7 promotor] and plate out on minimal medium plates. Incubate the plates overnight at 37°C.
  • A transformation protocol is given in the 'Cloning' section of the protocol database.
2. Pick one colony and use this to inoculate 5 ml Medium A. Incubate the culture overnight at 37°C.
3. Add the overnight culture to 1 L medium A and incubate the culture at the appropriate temperature for induction until the absorbance at 600 nm is between 0.8 and 1.0.
4. Induce overexpression of the target protein by adding 1 to 5 ml IPTG (200 mM) to the medium and incubate the culture for a further 2 to 12 hours.
5. Centrifuge the cell suspension for 10 min at 4,500 rpm (Sorvall RCB4 rotor) or 6,000 rpm (Sorvall GSA rotor) at 4°C.
6. Store the cell pellet at -20°C when not immediately used.