Part:BBa_K389004:Experience

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Applications of BBa_K389004

Measurement protocol

• For the luciferase detection we used a [http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System], containing a Cell Culture Lysis Reagent, Luciferase Assay Substrate and Luciferase Assay Buffer

• Prepare reaction tubes with 10 µL of high salt buffer (1M K₂HPO₄, 20mM EDTA, pH 7.8)

• Add 90 µL sample, mix and freeze at -80 °C

• For the measurement thaw by placing the tubes in room temperature water

• Add 300 µL of freshly prepared lysis mix ( 1X Cell Culture Lysis Reagent, 1.25 mg/mL lysozyme, 2.5 mg/mL BSA, add water for desired volume)

• Mix and incubate the cells for 10 minutes at room temperature

• Prepare the Luciferase Assay Reagent, by adding 10 mL of Luciferase Assay Buffer to the vial containing the Luciferase Assay Substrate

• Fill each well of a white, flat bottom 96 well microtiter plate with 20 µL of cell lysate

• For the detection of luciferase use a plate reading luminometer with injector for the Luciferase Assay Reagent and following settings (we used a Promega GloMax®-Multi Detection System with dual injector):
  • Injection volume of Luciferase Assay Reagent: 100 µL
  • Delay: 20 secs
  • Integration: 3 secs

User Reviews

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