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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, lysed by sonification

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.

Graph 1. Luminescence normalized for cell density (OD=1) at the time of sample preparation.