BISC 219/2009: Mod 3 Leaf Extract Preparation

Leaf Extract Preparation for the Spectrophotometric GUS activity assay using PNPG as substrate

You will need to prepare crude leaf extracts to measure the GUS activity spectrophotometrically in your putative transformants. Each pair of students should prepare extracts of four putative transformants and one of the non-transformed tobacco plants for use as a negative control.

For each plant that you are going to analyze, complete steps 1-4:

1. Using the top of a microfuge tube labeled with the plant #, punch 5 disks of young leaf tissue from the same plant. Place the leaf tissue you collected from the first plant in a pre-chilled mortar and pestle. Add 2 ml of cold extraction solution and a pinch of grinding sand. Grind until the tissue is thoroughly homogenized. This step is key so please make sure you do not hurry through this part.
2. Transfer all the homogenate in the mortar and pestle, using a Pasteur pipet into the labeled microfuge tube that you used to punch out the leaf disks for that plant. Store that tube of homogenate on ice or in the refrigerated centrifuge. Wash the mortar and pestle thoroughly in the sink and dry it so you do not cross-contaminate the extracts. Begin step 1 using the next plant.
3. Centrifuge all your extracts at 40 C for 15 minutes to clarify the homogenate.
4. Carefully transfer the clear supernatants, using either a Pasteur pipet or a micropipet into clean labeled tubes and store them on ice.

Leaf Extract Preparation
Spectrophotometric Assay for GUS activity
Calculations
GUS Activity Assay by Histochemistry
Structural Evidence for Transgenic Plants