Difference between revisions of "Expression of SeMet labeled proteins"

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1475 ml ddH<sub>2</sub>O
 
1475 ml ddH<sub>2</sub>O
 +
 
400 ml M63 stock salts
 
400 ml M63 stock salts
20 ml of solution 1
+
 
 +
20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=Expression_of_SeMet_labeled_proteins&action=submit#solution_1: solution 1]
 +
 
 
20 ml of solution 2
 
20 ml of solution 2
 +
 
20 ml of solution 3
 
20 ml of solution 3
 +
 
20 ml of solution 4  
 
20 ml of solution 4  
 +
 
20 ml of solution 5
 
20 ml of solution 5
 +
 
2 ml of 1M MgSO<sub>4</sub> stock
 
2 ml of 1M MgSO<sub>4</sub> stock
 +
 
20 ml of 20% glucose stock
 
20 ml of 20% glucose stock
 +
 
8 ml of ampicillin stock (100 mg/ml)
 
8 ml of ampicillin stock (100 mg/ml)
 +
 
10 ml of L-SeMet stock (10 mg/ml)
 
10 ml of L-SeMet stock (10 mg/ml)
 +
 
8 ml seed culture
 
8 ml seed culture
  
aliquote the 2 liter solution into 8X 250 ml  
+
aliquote the 2 liter solution into 8X 250 ml
 +
 
grow at 37°C over night until OD600=0.400
 
grow at 37°C over night until OD600=0.400
induce protein expression  
+
 
 +
induce protein expression
 +
 
harvest cells 20 hours later
 
harvest cells 20 hours later
 +
  
 
== M63 stock solution: ==
 
== M63 stock solution: ==
Line 25: Line 40:
  
 
15 g KH<sub>2</sub>PO<sub>4</sub>
 
15 g KH<sub>2</sub>PO<sub>4</sub>
 +
 
45.6 g K<sub>2</sub>HPO<sub>4</sub>
 
45.6 g K<sub>2</sub>HPO<sub>4</sub>
 +
 
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
 
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
 +
 
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
 
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
 +
  
 
== solution 1: ==
 
== solution 1: ==
  
for 50 ml (in ddH2O)
+
for 50 ml (in ddH<sub>2</sub>O)
  
 
add 200 mg of the following amino acids:
 
add 200 mg of the following amino acids:

Revision as of 20:40, 25 April 2008

Inoculate 2 l of the following media with 8 ml of seed culture (grown for 4 hours at 37°C in 2xYT):

1475 ml ddH2O

400 ml M63 stock salts

20 ml of solution 1

20 ml of solution 2

20 ml of solution 3

20 ml of solution 4

20 ml of solution 5

2 ml of 1M MgSO4 stock

20 ml of 20% glucose stock

8 ml of ampicillin stock (100 mg/ml)

10 ml of L-SeMet stock (10 mg/ml)

8 ml seed culture

aliquote the 2 liter solution into 8X 250 ml

grow at 37°C over night until OD600=0.400

induce protein expression

harvest cells 20 hours later


M63 stock solution:

for 1 liter (in ddH2O)

15 g KH2PO4

45.6 g K2HPO4

10 g (NH4)2SO4

2.5 ml FeSO4 at 2.5 mg/ml


solution 1:

for 50 ml (in ddH2O)

add 200 mg of the following amino acids: A, R, G, Q, H, I, L, K, P, S, T, V

pH should be adjusted to 7-7.5 with 1 M KOH

solution 2:

for 50 ml (in ddH2O)

add 200 mg of the following amino acids: N, D, C, E

pH should be adjusted to 7-7.5 with 1 M KOH

solution 3:

for 50 ml

200 mg phenylalanine 100 mg tryptophane 200 mg tyrosine 20 mg p-aminobenzoic acid 20 mg p-hydrobenzoic acid

dissolve in 50 ml 0.1 M KOH

solution 4:

for 50 ml

200 mg hypoxanthine 200 mg uracil

dissolve in 50 ml DMSO

solution 5:

for 500 ml (in ddH2O)

100 mg biotin 100 mg nicotinamide 10 mg riboflavin 100 mg thiamine