In light of the GFP results from yesterday, Reshma and Jason agreed with me that I should try pulsing the cells in PBS. I will perform this today as the last experiment of the summer. If this turns out well, I will plan on doing a J45120/J45181 time course in the same manner as soon as I get back from Pittsburgh. If this does not turn out well, we will go back to the drawing board.
Development of Protocol for GFP PBS experiment
1. Add the following in 2 2-L flasks:
- 275 mL LB + Antibiotic
- 27.5 uL grown I7100 or J45996 culture
2. Grow the cultures up for 20 mins at 220 RPM at 37C
3. Lower the RPM for the cultures to 110 RPM at 37C. The start of this step is Time Zero
4. After x hours (for x=6 (6:04) to x=12 (12:04), extract 25 mL of each culture and place into falcon tubes. Spin them down in the big centrifuge at 4C at 3800 RPM for 20 mins. Also, run 3 200 uL samples from each culture on the plate reader.
5. After centrifuging resuspend the cells in 25 mL PBS and add .5 mL .1 M salicylic acid. Then, transfer it to a 125-mL flask and shake it with the 2-L flasks for 3 hours.
6. At hour x+3 (9:04+ to 3:04+) for hour x, remove 3 200 uL aliquots of each culture from the falcon tubes and run them on the plate reader.
1. Clean up lab area- DONE (SORT OF)
2. Prepare presentation for lab meeting
3. Ask Austin about presenting early- DONE
4. Tell Lily that I'm going out of town but would like to be updated on the GC status- DONE
5. Ask grad students whether I should put the resuspended cells at RT, 30C, or 37C- DONE