Aug 17, 2016
GUS Solution
- Greg Reeves1
- 1University of Cambridge
- OpenPlant Project
Protocol Citation: Greg Reeves 2016. GUS Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.fdibi4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 21, 2016
Last Modified: March 28, 2018
Protocol Integer ID: 3210
Abstract
The GUS reporter system (GUS: β-glucuronidase) is a reporter gene system, particularly useful in plant molecular biology[1] and microbiology.[2]Several kinds of GUS reporter gene assay are available, depending on the substrate used. The term GUS staining refers to the most common of these, a histochemical technique. (Source: https://en.wikipedia.org/wiki/GUS_reporter_system)
Guidelines
- This GUS staining solution recipe is for a volume of 500mL.
- The solution can be partitioned into smaller tubes and stored long term at -20oC in the dark, as the GUS staining solution is light-sensitive.
Safety warnings
Note: The staining solution contains toxic chemicals.
https://en.wikipedia.org/wiki/Potassium_ferrocyanide
https://en.wikipedia.org/wiki/Potassium_ferricyanide
0.5M EDTA (pH 8.0)
1 mL
Note
EDTA will need to be pH adjusted with NaOH (Sodium hydroxide) until it reaches a pH of 8.0 in order to dissolve fully in deionised water. To make a 0.5M solution of EDTA, add 93.3g into 500mL of deionised water. Adjust pH to 8.0 with NaOH.
0.1M Potassium ferrocyanide
10 mL
Note
Both Potassium ferrocyanide (II) and Potassium ferricyanide (III) solutions should be made fresh each time a GUS staining solution is made. A higher concentration of Potassium ferro/ferricyanide (usually between 0.5mM and 10mM) constricts the movement of GUS between cells.
To make a 0.1M solution of Potassium ferrocyanide (II) add 0.42g into 10mL of deionised water.
To make a 0.1M solution of Potassium ferricyanide (III) add 0.33g into 10mL of deionised water.
0.1M Potassium ferricyanide
10 mL
1M Sodium phosphate buffer (pH 7.0)
50 mL
Note
The sodium phosphate buffer is formed from two sodium compounds (NaH2PO4, MW=137.99g/mol; and Na2HPO4, MW=141.96g/mol). These two compounds should be made up as two separate stock solutions and added together when making the sodium phosphate buffer for the GUS solution in this manner:
--To make a 1M solution of NaH2PO4, add 13.8g into 100mL of deionised water.
--To make a 1M solution of Na2HPO4, add 28.4g into 200mL of deionised water.
-To make 50mL of a 1M solution of sodium phosphate buffer, add 19.5mL of the 1M NaH2PO4 solution and 30.5mL of the 1M Na2HPO4 solution together. The pH of this 50mL solution should be at 7.0, so adjusting the pH should not be necessary.
X-Gluc
500 mg
dH20 up to 500mL
Note
After completion filter sterilze with 0.22um membrane. Aliquots can be frozen at -20oC prior to use.