Difference between revisions of "Expression of SeMet labeled proteins"

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(New page: 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 ...)
 
Line 2: Line 2:
 
culture (grown for 4 hours at 37°C in 2xYT):
 
culture (grown for 4 hours at 37°C in 2xYT):
  
1475 ml ddH2O
+
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 solution 1
Line 20: Line 20:
 
harvest cells 20 hours later
 
harvest cells 20 hours later
  
M63 stock solution:
+
== M63 stock solution: ==
  
 
for 1 liter (in ddH2O)
 
for 1 liter (in ddH2O)
  
15 g KH2PO4
+
15 g KH<sub>2</sub>PO<sub>4</sub>
45.6 g K2HPO4
+
45.6 g K<sub>2</sub>HPO<sub>4</sub>
10 g (NH4)2SO4
+
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
2.5 ml FeSO4 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 ddH2O)
Line 36: Line 36:
 
A, R, G, Q, H, I, L, K, P, S, T, V
 
A, R, G, Q, H, I, L, K, P, S, T, V
  
pH should be adjusted to 7-7.5 with 1M KOH
+
pH should be adjusted to 7-7.5 with 1 M KOH
  
solution 2:
+
== solution 2: ==
  
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:
 
N, D, C, E
 
N, D, C, E
  
pH should be adjusted to 7-7.5 with 1M KOH
+
pH should be adjusted to 7-7.5 with 1 M KOH
  
solution 3:
+
== solution 3: ==
  
 
for 50 ml
 
for 50 ml
Line 59: Line 59:
 
dissolve in 50 ml 0.1 M KOH
 
dissolve in 50 ml 0.1 M KOH
  
solution 4:
+
== solution 4: ==
  
 
for 50 ml
 
for 50 ml
Line 68: Line 68:
 
dissolve in 50 ml DMSO
 
dissolve in 50 ml DMSO
  
solution 5:  
+
== solution 5: ==
  
for 500 ml (in ddH2O)
+
for 500 ml (in ddH<sub>2</sub>O)
  
 
100 mg biotin
 
100 mg biotin

Revision as of 20:36, 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