Team:Freiburg/Protocols/LUC
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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 | 10µl |
DTT | 1 M | 200 µl |
ATP | 100 mM | 12.5 µl |
BSA | - | 4 mg |
CoA | 30 mM | 0.6 µl |
Dilution Reagent
Component | stock concentration | amount in 50 µl reaction [µl] |
---|---|---|
Master mix | - | 17.68 |
Lysate | - | 22.5 |
DNA | at least 217 ng/µl | 3250 - 5000 ng |
H2O (nuclease free) | - | up to 50 µl |
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
- add creatine phosphokinase last to mastermix
- work quickly, sterile and as nuclease free as possible (on ice)
- add DNA last to start reactions simultaneously
- amount of lysate used can be varied (here 45%)
- the used chemicals should have a high grade of purity
- Certain counterions like Chlor and Sodium should be avoided
- feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly
Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany