×
Please leave your comments or questions
 

Firefly luciferase assay

Author: Gal Haimovich, updated date: , view: 224, 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'.



Comment
Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.