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Latest revision as of 07:11, 20 November 2015

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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 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