IGEM:MIT/2006/Notebook/2007-6-14

Time Course of J45181

We will go ahead with this. The 10 mL overnight cultures of J45181 smelled with both the old and new sodium salicylate solution added.

1. Take a 250 mL flask. Added 120 mL LB AMP/TET. Added 2.4 mL of .1 M sodium salicylate. Added 60 uL of 5 mL overnight culture of J45181.

2. Split the flask into 6 20 mL cultures (in 250 mL flasks).

3. Remove 1 flask every 4 hours for 20 hours (including time zero) (10 AM, 2 PM, 6 PM, 10 PM, 2 AM, and 6 AM) and immediately follow GC sample extraction protocol.

Settings for shaker:

110 RPM, 37 degrees C

GC Sample Extraction Protocol

1. Rinse separatory funnel 3 times with 10 mL n-heptane between samples (5 times for the time zero sample). *NOTE- actually washed 2 times with 8 mL n-heptane for latter 4 samples

2. Pour 20 mL sample into separatory funnel.

3. Add 2 mL n-heptane.

4. Invert separatory funnel 24 times, releasing the top every eight inversions.

5. Wait 8 minutes for the solution in the funnel to phase separate.

6. Release the bottom part of the solution, leaving only the nonpolar, clear liquid in the funnel.

7. Remove 200 uL of the liquid. Place it in 2 mL tube. That is your sample, which must be stored at 4 degrees C.

8. Repeat for each sample.

Other Stuff to Do

1. Perform 3 minipreps each of the 15 mL LCs of J45181 and J45400- DONE

2. Obtain more n-heptane from either the Endy or Knight lab- WORKING ON IT

3. Contact Li Li about GC tomorrow- DONE

4. Perform ES digest of J45181 minipreps (gave up on XP for now), XP digest of J45400 and J45320 digests- DONE

5. Run gels on digests above (2 regular and 1 gel extraction gel each)- DONE (ALL LOOK GREAT EXCEPT FOR GEL EXTRACT BAND OF J45400!!! YEH!!!)

6. Gel extract J45181 and J45320 bands- DONE

7. Ask Isadora to order another liter of n-heptane- DONE (WILL BE IN MONDAY)