TUDelft/19 September 2008
From 2008.igem.org
(New page: {{Template:TUDelftiGEM2008}} {{Template:TUDelftiGEM2008_menu_home}} {{Template:TUDelftiGEM2008_calendar}} =September 19th 2008= ==Luciferase Assay== Today we've done our first luciferase...) |
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Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control). | Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control). | ||
===Samples=== | ===Samples=== | ||
- | A: 12, Growing overnight with induction at room temperature, lysed by sonification | + | A: 12, Growing overnight with induction at room temperature, lysed by sonification<br> |
- | B: 34, Growing overnight with induction at room temperature, lysed by sonification | + | B: 34, Growing overnight with induction at room temperature, lysed by sonification<br> |
- | C: 12, Growing overnight with induction at 37oC, lysed by sonification | + | C: 12, Growing overnight with induction at 37oC, lysed by sonification<br> |
- | D: 34, Growing overnight with induction at 37oC, lysed by sonification | + | D: 34, Growing overnight with induction at 37oC, lysed by sonification<br> |
- | E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification | + | E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification<br> |
- | F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification | + | F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification<br> |
- | G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification | + | G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification<br> |
- | H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification | + | H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification<br> |
- | I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer | + | I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer<br> |
- | J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer | + | J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer<br> |
- | K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness. | + | K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness.<br> |
===Protocol=== | ===Protocol=== |
Revision as of 10:59, 22 September 2008
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Contents |
September 19th 2008
Luciferase Assay
Today we've done our first luciferase assay. The samples we had in the end were either K115034 (34) or K115012 (12, control).
Samples
A: 12, Growing overnight with induction at room temperature, lysed by sonification
B: 34, Growing overnight with induction at room temperature, lysed by sonification
C: 12, Growing overnight with induction at 37oC, lysed by sonification
D: 34, Growing overnight with induction at 37oC, lysed by sonification
E: 12, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
F: 34, Growing overnight without induction at 37oC, induce next day for 5 hours at room temperature, lysed by sonification
G: 12, Growing 2 times overnight with induction at room temperature, lysed by sonification
H: 34, Growing 2 times overnight with induction at room temperature, lysed by sonification
I: 12, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
J: 34, Growing 2 times overnight with induction at room temperature, lysed by kit buffer
K: 12, Growing o/n at 37oC without induction, to test R0080 for leakiness.
Protocol
The protocol used in the case of most samples, when lysed by sonification was: 1. Grow as indicated above and induce immediately, unless stated otherwise 2. Pellet 3 ml of cells 3. Resuspend in 100 ul 15 mM Tris HCl 4. Destroy by sonification (3 * 15 s) 5. Freeze at -20 6. Add 20 ul of lysed cell sample to a well 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)
If lysis by kit buffer (Promega) was used, the protocol became: 1. Grow as indicated above and induce immediately, unless stated otherwise 2. Pellet 3 ml of cells 3. Resuspend in 100 ul 1X lysis buffer 4. Leave at room temperature for 15-30 minutes 5. Freeze at -20 6. Add 20 ul of lysed cell sample to a well 7. Measure by automatic dispension of 100 ul of Luciferase Assay buffer (Promega)
Results
After a lot of fine tuning of the luminometer, the luminescence values were obtained as shown in graph 1.