IGEM:MIT/2006/Notebook/2007-8-23

J45200/J45250 3-Hour Pulse experiment

Protocol

Protocol for 3-hour pulse:

1. Add the following in a 2-L flask:

• 275 mL LB AMP/TET

• 27.5 uL grown J45120 culture

2. Add the following in a 2-L baffled flask:

• 275 mL LB AMP/TET

• 27.5 uL grown J45181 culture

3. Grow both cultures at 110 RPM at 37C, starting J45200 20 minutes before J45250. Once the culture is put at 110 RPM and 37C, it is Time Zero

4. After x hours (for x=4 to x=12), extract 25 mL culture from master culture, pour into 125-mL flask, and add 13.6 uL to it for both J45200 and J45250 (grow up at 110 RPM)

5. At hour x+3 for hour x, GC extract culture

OD600s

Before Isoamyl Alcohol Addition

J45200

4 hours- 0.06

5 hours- 0.18

6 hours- 0.54

7 hours-

8 hours-

9 hours-

10 hours-

11 hours-

12 hours-

J45250

4 hours- 0.08

5 hours- 0.26

6 hours-

7 hours-

8 hours-

9 hours-

10 hours-

11 hours-

12 hours-

Other Things to Do

1. Swing by Stephanopoulos lab to see status of the GC- DONE (bad news: delayed, needs until Saturday to set it up; good news: nobody is set up to use it, so I should be able to use it all of next week)

2. At 4:40, dilute the J45995, J45996, I7100, and B0015 cultures 1:250