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Team:Cambridge/Protocols/beta-galactosidaseassay
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Revision as of 10:46, 3 September 2012
β-galactosidase Assay
Theory
- X-Gal itself is colourless and undetectable in the spectrophotometer.
- X-Gal is cleaved by β-galactosidase, forming 5-bromo-4-chloro-3-hydroxyindole.
- This dimerises to form 5,5'-dibromo-4,4'-dichloro-indigo. This can be detected as a considerable change in the colour of the solution.
- The precise shade of the resultant solution can therefore be used to quantify the amount of β-galactosidase reporter being produced.
Protocol
- Take suspension of cells grown up overnight and dilute 200 times in appropriate growth medium (for example LB broth for e.coli). If genetic construct can be selected for by an antibiotic, include this in the growth medium according to needs - this prevents loss of the plasmid during growth.
- Now add the following to each of your eppindorf tubes:
| Reagent | Volume (µl) |
| X-Gal (10mg/ml) | 20 |
| Substance of interest (e.g. Fluoride) (0.5 M) | 1 for each mM desired in final solution |
| Cell suspension | Make up to 500 μl final volume |
- Leave tubes overnight in 37 °C incubator.
- Tubes with greatest β-galactosidase activity will have greatest blue colouration.
