Team:Freiburg/Protocols/LUC
Protocol Cell-free expression
How to perform a Luciferase assay
material: Luciferase Reaction Mix, Dilution Reagent, Luciferse DNA, Cell-free expression Mix
duration: Preparation 20 min + running time
Luciferase Reaction Reagent
| Component | stock concentration | amount of stock for 2 ml |
|---|---|---|
| D-Luciferin | 100 mM | 10 µl |
| DTT | 1 M | 200 µl |
| ATP | 100 mM | 12.5 µl |
| BSA | - | 4 mg |
| CoA | 30 mM | 0.6 µl |
Dilution Reagent
| Component | end concentration |
|---|---|
| TRIS-phosphate, pH 7.8 | 25 M |
| DTT | 2 mM |
| EDTA | 2 mM |
| Glycerol | 10% |
| TritonX100 | 1% |
| BSA | 1 mg/ml |
- Both solutions can be stored at -20°C
Implementation
- To characterize a reaction, the luciferase plasmid was added to the expression mix and the expression is run using desired conditions (standard: 2 h at 37°C, no shaking). Afterwards, the reaction is stopped by putting it on ice. Inside a well of a 96-well microplate, 20 µl of the reaction are mixed with 20 µl of dilution reagent, creating a 1:2 dilution. If a 1:4 dilution is demanded, 20 µl of the first dilution are added to another 20 µl dilution reagent, otherwise they are discard-ed. 50 µl of the luciferase assay reagent is added to the reaction only seconds before the measurement is started. The microplate reader is set to perform a full spectrum emission scan (400 - 700 nm) of each well. Because of the kinetic temperature dependency of luciferase, it was minded to perform every scan at 25°C.
Remarks
- The Reaction Reagent tends to get contaminated so regular preparation is necessary
