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Expression of SeMet labeled proteins: Difference between revisions
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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 ...) |
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| 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 | 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 | 15 g KH<sub>2</sub>PO<sub>4</sub> | ||
45.6 g | 45.6 g K<sub>2</sub>HPO<sub>4</sub> | ||
10 g ( | 10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> | ||
2.5 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 | pH should be adjusted to 7-7.5 with 1 M KOH | ||
solution 2: | == solution 2: == | ||
for 50 ml (in | 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 | 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 | for 500 ml (in ddH<sub>2</sub>O) | ||
100 mg biotin | 100 mg biotin | ||