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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

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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

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