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)- DONE

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

9. Sign up for plate reader Wednesday- DONE

10. Run GC samples- DO IT EARLY TOMORROW

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

Protocol for Next Time

1. At each time point, aliquot 20 mL cultures (J45120 and J45181) into falcon tubes and remove .5 mL to take an OD600 measurement.

2. Incubate culture for 10 minutes on ice.

3. Centrifuge the samples at 4C for 20 mins at 3800 RPM.

4. Discard the supernatants. Store the pellets at -80C until the last time point.

5. Once all time points are collected, resuspend pellets in 10 mL Tris buffer (10 mM Tris, 100 mM NaCl, pH 8).

6. Centrifuge the samples at 4C for 20 mins at 3800 RPM.

7. Discard supernatants.

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

9. Add 19 mL LB AMP/TET to samples.

(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 samples.

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

12. GC extract the samples.