BISC 219/2009: Mod 3 GUS Activity Assay by Histochemistry

GUS Activity Assay by Histochemistry

The goal of this procedure is to obtain additional evidence that your cloned plants are transgenic through an additional β-glucuronidase activity assay. This assay is only semi-quantitative, but it allows testing whole leaf tissue directly.

1. Punch one leaf disk from each of the same plants on which you performed the other enzyme activity assay (1 control and 4 putative transformants). Place each disk into the well of a microtiter plate containing the GUS assay mixture (X-glucoronide= 5-bromo 4-chloro 3-indolyl beta-D glucoronide in DMSO) Note the well number for each sample in your notebook.
2. Place the microtiter dish under vacuum to remove the air trapped within the tissue.
3. Wrap the dish in saran wrap and place in the 37 C incubator for 24 hours.
4. Stop the assay and extract the chlorophyll by removing the assay solution and replacing it with 75% ETOH. It will take several hours for the chlorophyll to be extracted from the tissue thus making the blue GUS enzyme reaction product readily visible.
5. After a 24 reaction period score each disk for intensity of blue color using the following relative scale: 3+ very blue; 2+ blue; 1+ slightly blue; 0 not blue

Gus Activity Histochemical Stain: X-glucoronide (5_Bromo4 chloro3indolyl-beta-D glucoronide in DMSO), 1 M sodium phosphate (pH 7), 0.05% Triton X100.

Assaying the Transgenic Plants (HOME)
Leaf Extract Preparation
Spectrophotometric Assay for GUS activity
Calculations
Structural Evidence for Transgenic Plants