Lecture Topics

• GUS staining

Experimental Procedures

• Staining of transgenic plants

Overview of Procedures

Each team will be given plates of seedlings representing 2 lines of GUS transgenic plants and both positive and negative controls.
Seedlings will be stained with X-GLUC in 12 or 24 well plates.

GUS Staining

• 200 mM NaPO4, pH 7.0
  1. Weigh out 8.66 g Na2HPO4 and 4.7 g NaH2PO4.
  2. Bring total volume to 500 mL with Milli-Q water.
  3. Check pH with paper.
  4. Autoclave to sterilize.
  
  
• X-GLUC Buffer
  
  125 ml 200 mM NaPO4, pH 7.0
  • 0.413 ml 10% K3[Fe(CN6)], or 1.25 ml of 0.1 M
  • 0.527 ml 10% K4[Fe(CN6)]x3H2O, or 1.25 ml 0.1 M
  • 5 ml 0.5 M EDTA
  • 0.25 ml 10% Triton X-100
  • ml water
  • Filter sterilize
  • Store at 4°C, covered with foil (CN is light sensitive)
  
  
• X-GLUC Staining Solution
  
  • Let the X-GLUC substrate sit at room temperature before mixing the staining solution.
    
  • Weigh out 1 mg X-GLUC per ml of staining solution.
    (Note: X-GLUC must be dissolved in 200 µL DMSO or DMF first--it will not dissolve in water)
    
  • Dilute to proper volume with X-GLUC buffer.
  
  
• Staining Protocol
  1. Carefully remove the plant tissue and place into one well of a 12 or 24 well tissue culture plate containing 1-2 mL (enough to completely cover tissue) of X-GLUC staining solution.
  2. Vacuum infiltrate for 10 mins at 600 mmHg at room temperature.
  3. After releasing the vacuum slowly, allow tissue to incubate in the staining solution overnight to several days at 37°C; cover the plate with foil or place in a dark chamber.

     YOU WILL HAVE TO COME IN THE DAY BEFORE LAB # 6 TO STOP THE STAINING REACTION AND CLEAR THE CHLOROPHYLL.

  4. Remove the staining solution.
  5. Add 2 mL RO-water to stop the staining reaction.
  6. Let sit for 2-5 mins, then remove the water.
  7. Immerse the tissue in 50% ethanol overnight to bleach the tissue of chlorophyll.
     *Alternatively, immerse the tissue in 100% ethanol for 15-30 min. and rinse several times with ethanol until the green tissue turns white.

  ---

  Copyright, Acknowledgements, and Intended Use
  Created by B. Beason (bbeason@rice.edu), Rice University, 23 July 2003
  Updated 4 November 2004