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.'' 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
 * 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