IGEM:MIT/2006/Notebook/2007-8-20

Things to Do

1. Develop protocol for lysing cells (I see B-PER II solution on his bench)- DONE (have a protocol to follow, see below)

2. Call for a meeting tomorrow- DONE

3. Discuss conference logistics with Veena and Drew

4. Do lysis protocol/wintergreen time course for Hour 13 (2:58, 3:18), 14 (3:58, 4:18), and 15 (4:58, 5:18).

5. Organize results from plate reader experiment and send it out to advisors- DONE

6. Collect materials for lysis step- DONE

7. Make Tris buffer solution- DONE

8. Ask Isadora about ordering more heptane- DONE

Protocol for Time Course

1. At each time point, aliquot 20 mL culture into falcon tube and remove .5 mL to take an OD600 measurement.

• OD600s: J45120 13 hours- 2.40; J45181 13 hours- 2.36; J45120 14 hours- 2.48; J45181 14 hours- 2.38; J45120 15 hours- 2.54; J45181 15 hours- 2.40

2. Put Tris buffer (10 mM Tris, 100 mM NaCl, pH 8) at 4C.

3. Incubate culture for 10 minutes on ice.

4. Centrifuge the sample at 4C for 4 mins at 13000 RPM (balancing it with a falcon tube containing 20-mL water).

5. Remove supernatant.

6. Resuspend sample pellet in 10 mL Tris buffer.

7. Repeat steps 4 and 5.

8. Resuspend cells in 1 mL B-PER II.

9. Add 19 mL water to sample.

(optional: add 200uL of 100mM protease inhibitor to the cell lysate. to prepare 100mM PMSF (MW = 174.19), add .1742 g to 10mL isopropanol) *we added .03 g to 1.5mL isopropanol to make 1.5mL of 100mM PMSF

10. Add .4 mL .1 M salicylic acid to sample.

11. Let sample incubate for 2 hours at room temperature after addition of salicylic acid.

12. GC extract the sample.

3 NOTES: J45181 was delayed by 10 mins to :28 of each hour; Protease inhibitor added late to J45120 13 hour point; Cells spinned down at 1300 RPM, not 13000 RPM