Silver: RNA Export Assay

(adapted fr. E. Lei, Feb. 2002)

1. Grow 5mL of culture to early log phase (1 x 10⁷ to 2 x 10⁷ cells/mL).
   • Temperature shift if desired – use waterbath for shorter temperature shift or warm room for longer (4h or so)
2. Fix by adding culture to 15mL Falcon tube containing 350µL 37% formaldehyde (inside the chemical hood, dispose the waste in the proper waste jug inside the hood!).
   • Mix well.
3. Incubate 1 hr with agitation at the temperature at which the cells were grown.
   • For zero timepoints, I sometimes fix cells at the high temperature.
4. Spin down cells for 2 min at 2000 x g.
5. Wash twice with 1mL 0.1M phosphate buffer (pH 6.5)
6. Flash spin to pellet cells and discard sup, followed by a single wash with 1mL P sol’n.
7. Discard waste via aspiration.
8. Resuspend pellet in 100µL to 1mL P sol’n to desired cell density. (A culture at 1 x 10⁷ cells/mL can be resuspended in 200µL)