IGEM:MIT/2006/Notebook/2007-8-23: Difference between revisions
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Revision as of 17:38, 13 October 2007
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 (Repeat 10/13)
-Start at 12:07 for 200 and 12:27 for 250
Before Addition of Isoamyl Alcohol
J45200
2- 0.00
3- 0.00
4- 0.04
5- 0.16
6- 0.42
7- 0.98
J45250
2- 0.00
3- 0.00
4- 0.06
5- 0.18
6- 0.52
7- 1.10
After Addition of Isoamyl Alcohol
J45200
5- 0.00
6- 0.04
7- 0.16
8- 0.60
9-
10-
J45250
5- 0.02
6- 0.04
7- 0.18
8- 1.02
9-
10-
Side experiment (10/13)
-Make 50 mL, 250 uL culture of 181 AT3, 250 AC3 and grow at 220 RPM for 20 mins (12:07)
-Add isoamyl alcohol and salicylic acid (standard amounts) and split cultures
-GC extract one in exponential (OD600 = 0.32, WORRIED) and one in stationary phase
Other Side experiment (10/13)
-Took J45200 after 7 hrs and added 2.28 uL isoamyl acetate to 20 mL culture (100 ppm IAc) to get a data pt for extraction efficiency
OD600s (Repeat 9/29)
Before Isoamyl Alcohol Addition
J45200
4 hours- 0.12
5 hours- 0.34
6 hours- 0.76
7 hours- 1.30
8 hours- 1.92
9 hours- 2.14
10 hours- 2.44
11 hours- 2.52
J45250
4 hours- 0.18
5 hours- 0.50
6 hours- 1.14
7 hours- 1.58
8 hours- 1.92
9 hours- 2.06
10 hours- 2.24
11 hours- 2.34
After Isoamyl Alcohol Addition
J45200
7 hours- 0.46
8 hours- 1.28
9 hours- 1.48
10 hours- 1.68
11 hours- 1.92
12 hours- 2.12
13 hours- 2.40
14 hours- 2.68
J45250
7 hours- 1.00
8 hours- 1.26
9 hours- 1.74
10 hours- 1.92
11 hours- 1.98
12 hours- 2.10
13 hours- 2.26
14 hours- 2.44
OD600s
Before Isoamyl Alcohol Addition
J45200
4 hours- 0.06
5 hours- 0.18
6 hours- 0.54
7 hours- 1.02
8 hours- 1.60
9 hours- 2.04
10 hours- 2.32
11 hours- 2.54
12 hours- 2.76
J45250
4 hours- 0.08
5 hours- 0.26
6 hours- 0.66
7 hours- 1.44
8 hours- 1.70
9 hours- 1.84
10 hours- 2.08
11 hours- 2.16
12 hours- 2.30
After Isoamyl Alcohol Addition
J45200
7 hours- 0.34
8 hours- 1.08
9 hours- 1.34
10 hours- 1.58
11 hours- 1.76
12 hours- 2.06
13 hours- 2.40
14 hours- 2.64
15 hours- 2.88
J45250
7 hours- 0.46
8 hours- 1.34
9 hours- 1.52
10 hours- 1.82
11 hours- 1.88
12 hours- 2.00
13 hours- 2.16
14 hours- 2.24
15 hours- 2.42
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- SCRAP THIS (NOT ENOUGH FLASKS)
3. Ask grad students about next step in wintergreen curve production- DONE (go with gut instinct)
