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Expression of SeMet labeled proteins: Difference between revisions
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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: == | ||
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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 | 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: | ||