Team:Cambridge/Protocols/beta-galactosidaseassay

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(Theory)
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===Theory===
===Theory===
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[[File:x-galreaction.jpg|right|500px|thumb|Chemical reaction by which x-gal can be detected]]
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[[File:x-galreaction.jpg|right|500px|thumb|Chemical reaction by which X-Gal produces a coloured solution in the presence of β-galactosidase.]]
* X-Gal itself is colourless and undetectable in the spectrophotometer.
* X-Gal itself is colourless and undetectable in the spectrophotometer.

Revision as of 10:45, 16 August 2012

β-galactosidase Assay

Theory

Chemical reaction by which X-Gal produces a coloured solution in the presence of β-galactosidase.
  • 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:
ReagentVolume (µl)
X-Gal (10mg/ml)20
Substance of interest (e.g. Fluoride) (0.5 M)1 for each mM desired in final solution
Cell suspensionMake up to 500 μl
  • Leave tubes overnight in 37 °C incubator.
  • Tubes with most β-galactosidase activity will be most blue. This can be quantified using a spectrophotometer at wavelength 420 nm.