H-Leucine into E. coli Proteins

Grow a 25 ml culture of wild type E. coli to a cell density of 45 Klett units (this is early log phase) on Ozeki s minimal medium (27) in a shaking water bath adjusted to 30°C. 

Prepare 0.5-1.0ml of a H-leucine solution containing 25/ag leucine per 0.1 ml at a specific activity of 1 /ac//amole. 

Dissolve 10 mg rifampicin (rifamycin SV) in 1.0 ml dimethyl sulfoxide. Prepare also a 5% (w/v) solution of trichloroacetic acid (TCA). 3-107. Place exactly 0.1 ml H-leucine solution (step 3-105) in a 125 ml Erlenmeyer flask and place the flask in the bath. 

When the culture reaches a cell density of 45 Klett units, transfer exactly 6.0 ml to the 125 ml flask and immediately begin timing the experiment using a stopwatch or digital laboratory timer. 

and 12 minutes after cells were added to the radioactive leucine solution, remove 0.2 ml samples and place them in ice cold 13 X 100 mm test tubes. Immediately after transfer, add 5 ml cold 5% TCA to the test tubes. 

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