LOCAL

Lab Protocols

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.