Materials 

                                           For 10 liter culture medium

1. 10X M9

NH₄Cl       20 g/l                                                          20g

     KH₂PO₄      60 g/l                                *1000ml        60g

     Na₂HPO₄     120 g/l                                                  120g

2. 10X MgSO₄．7H₂O    5 g/l                  *1000ml        5g

3. 40X FeSO₄．7H₂O     1 g/l                   *250ml        0.25g   4度C

4. 20X

     Roboflavin     0.02 g/l                             *500ml         0.01g

     Niacin amide   0.02 g/l                                                0.01g

5. 1000X

     Pyridoxin   1 g/l                                         *10ml          0.01g

Thiamin    1 g/l                                                              0.01g

6. 20X glucose   160 g/l                             *500ml             80g

7. 50X Stock I: Trp, Phe   4 mg/ml            *200ml         800mg

  40X Stock II: Asn, Asp, Ala, Arg, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr, Val  

                                          3.2 mg/ml           *250ml         800mg

  4X Stock III: Tyr   0.32 mg/ml                 *2500ml         800mg 

  40X Se-Met: 3.2 mg/ml                             *250ml         800mg 

Procedure 

Day1

1. Autoclave bottles (2L*1, 1L*3, 0.5L*2, 0.25L*4) and flask (0.5L*1).

2. Transform expression plasmid to E. coli strain B834.

Day2

1. Prepare a 3mL day culture consisting of 3mL LB media, 3uL antibiotics (1000x conc.), and a single E.coli colony. Grow at 37°C all day.

2. Prepare buffer and amino acid stock (0.22uL filtered).

3. Prepare a 200mL overnight culture (centrifuge day culture, suspend pellet by M9 buffer, transfer E.coli to 200 mL culture medium)

Day3

1. Incubate 1 liter culture medium with 20mL overnight culture and grow until an OD600=0.5-0.6 (~6-8 hours)

2. Induce with 1mL 1M IPTG (final concentration = 1mM).

3. Grow about 18-20 hours at 18°C

4. Collect cells as usual and proceed to purification steps.

Download this file :  http://hyuan.imb.sinica.edu.tw/protocol/Se-Met.doc