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Firefly luciferase assay

Author: Gal Haimovich, updated date: , view: 6, Q&A: 0
Tag: luciferase

Materials and Reagents


  1. TRIS-HCl pH7.5 1M

  2. MgCl2 1M

  3. ATP 200mM

  4. D-luciferin 10mM

  5. TRITON X-100 (10%)

  6. PBS x1

  7. 70% ethanol

  8. ddwater

  9. 12-well plate

  10. 96-well plate, black


Equipment

luminometer (veritas) in Adi Kimchi's lab

Procedure

  1. cutlure cells and treat approprietly.

  2. Wash cells once in PBSx1

  3. Add 100µl lysis buffer.  wait 2 minutes RT then use pipetor up&down to lyse all cells in well.

  4. Transfer 40µl x2 to black 96-well.

  5. prepare 50ml tube with ddwater, 50ml tube with 70% ethanol, empty 50ml tube (waste) and 15ml with luciferase reaction buffer wrapped in aluminium foil [prepare 100µl/well in 96 plate + 1ml extra].


reading results:

Start the machine, then start Veritas software.

go to "saved procedure --> luciferase --> galh"

click "options" --> choice injector 1" and choose wells. press "Apply choice"


Click "Prime" --> injector 1. follow instructions ans startthe priming. at the end, return the tube to its holder.

press "Start" - the reading will now take place.

after its done, press "Flush" and follow instructions (was once in water --> ethanol --> water). make sure "flush cycle" is set to '3'.



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