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.
- 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.
- Place the microtiter dish under vacuum to remove the air trapped within the tissue.
- Wrap the dish in saran wrap and place in the 37 C incubator for 24 hours.
- 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.
- 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
Structural Evidence for Transgenic Plants
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