M9 Method (Met Auxtroph)
NOTE: SeMet is TOXIC. The culture supernatant should be collected for disposal as hazardous waste. Fill the waste bottles to shoulder-height, not all the way to the screw cap. Use the Online Tag Program to generate hazardous waste tags as soon as you start generating the hazardous waste and affix the labels to the waste bottles immediately.
Refer to your lab-specific Standard Operating Procedure (SOP) for the proper way to work with SeMet. At a minimum you will need to use the appropriate Personal Protective Equipment (PPE) which includes the use of nitrile gloves, lab coat, and full-coverage shoes.
Start with expression plasmid transformed into Met auxotroph cells [i.e. E. coli B834 (DE3)].
Day 1
Start overnight culture from individual colony in LB media.
Day 2
Dilute 1:100 into an M9 semet O/N culture.
Day 3
Inoculate 2L of defined M9 media with 20 ml overnight culture. Incubate at 37C until OD600=0.4-0.6. Add IPTG to 1 mM and grow for an additional 5-15 hours at desired temperature.
M9 Medium 2L
5X M9 salts 400 ml
10% Glucose 200 ml
1M MgSO4 4 ml
1M CaCl2 200 ul
20ml 100X Kao Vitamins (Sigma, K3129,Kao and Michayluk Vitamin Solution)
10X soluble AA 200 ml
100X insoluble AA 20 ml
SeMet 100 mg
dH2O to 2L
Autoclave M9 salts and water. After flask has cooled add other ingredients
5X M9 Salts
Na2HPO4.7H2O 64 g
KH2PO4 15 g
NaCl 2.5 g
NH4Cl 5.0
dH20 1 L
10X soluble Amino Acids
Alanine 0.5g
Arginine 0.5g
Aspartic Acid 0.5g
Asparagine 0.5g
Cystine 0.5g
Glutamic Acid 0.5g
Glutamine 0.5g
Glycine 0.5g
Histidine 0.5g
Isoleucine 0.5g
Leucine 0.5 g
Lysine 0.5g
Phenylalanine 0.5g
Proline 0.5g
Serine 0.5g
Threonine 0.5g
Valine 0.5g
dH2O 1 L
100X amino acids
Tryptophan 0.5g
Tyrosine 0.5g
Dissolve in 10 ml of 2M HCl and slowly add dH2O to 100 ml. Filter sterilize and store at -20C.