Categorized: Microbiological Techniques
- Grow an overnight culture of H103 (wildtype) in 10 ml of minimal medium containing 1/20 normal amount of sulfate.
- Subculture the overnight 1/40 into fresh medium containing no sulfate.
- Grow to OD600 = 0.1-0.2.
- Add unlabelled methionine to 1 mM (0.1 ml of 0.1 M stock of autoclaved unlabelled methionine into 10 ml of cells) and 35S-methionine to a final concentration of 50 µCi/ml. Return cells to incubator.
- To monitor uptake of label, remove 1 ml samples at 30 minute intervals, filter cells through 0.45 micron filters, wash twice with 0.1 M LiCl, dry and count filters.