Herskowitz Lab Protocol
From : RKT 7/96


Bgal Liquid Assays

  1. Grow cells to log phase in liquid culture
  2. Check OD600 by diluting 0.5ml culture in 0.5ml media
  3. When reading many samples, use yellow tip on P200 to remix cells before each reading since they settle
  4. Read OD more than once. MINIMIZE PIPETTE ERRORS--INCLUDE A BLANK REACTION
  5. Put 1ml culture into an eppendorf (in triplicate)
  6. Spin 5minutes (to get hard pellet)
  7. Aspirate with P1000, (do not use the vacuum line)
  8. Wash in 1ml Z buffer (w/o BME)
  9. Eepellet
  10. Suspend in 150ul Z buffer (with BME -- 27ul/10ml)
  11. Add 50ml chloroform
  12. 20ml 0.1% SDS
  13. Vortex hard 15"
  14. Add 700ul prewarmed ONPG (1mg/ml in Z+BME)
  15. Time reaction at 30°C (20' to 3hr)
  16. Stop with 0.5ml 1M NaCO3
  17. Pellet 10'
  18. Read A420
  19. Calculate using the following formula:



Z Buffer:

Na2HPO4.7H2O 16.1g (8.5g anhydrous)
NaH2PO4.H2O 5.5g
KCL 0.75g
MgSO4.7H2O 0. 246g ---> 1L


Related protocols include a semi-quantitative filter assay and a semi-quantitative agarose overlay assay.


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