Herskowitz
Lab Protocol
From : RKT 7/96
Bgal Liquid Assays
- Grow cells to log phase in liquid culture
- Check OD600 by diluting 0.5ml culture in 0.5ml media
- When reading many samples, use yellow tip on P200 to remix cells before each reading since they settle
- Read OD more than once. MINIMIZE PIPETTE ERRORS--INCLUDE A BLANK REACTION
- Put 1ml culture into an eppendorf (in triplicate)
- Spin 5minutes (to get hard pellet)
- Aspirate with P1000, (do not use the vacuum line)
- Wash in 1ml Z buffer (w/o BME)
- Eepellet
- Suspend in 150ul Z buffer (with BME -- 27ul/10ml)
- Add 50ml chloroform
- 20ml 0.1% SDS
- Vortex hard 15"
- Add 700ul prewarmed ONPG (1mg/ml in Z+BME)
- Time reaction at 30°C (20' to 3hr)
- Stop with 0.5ml 1M NaCO3
- Pellet 10'
- Read A420
- 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.
