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. It is important not to contaminate the non-transgenic control with leaf material from the putative transgenics; therefore, make the extract of your non-transgenic control FIRST and use only the mortar and pestles that are kept separate and designated CONTROL.
For each plant (non-transgenic control and putative transgenics) that you are going to analyze, complete steps 1-4. Remember to do the control first and make sure that you use a mortar and pestle for the control that has NOT been used previously to make an extract from a putatative transgenic
- 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 1.5 ml (use your P1000 set to 750 microliters twice) of cold extraction solution and a tiny 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.
- 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 to make an identical extract of the next plant.
- Centrifuge all your extracts at 4degreesC for 15 minutes to clarify the homogenate.
- Carefully transfer only the clear supernatant, using either a Pasteur pipet or a micropipet into a set of clean labeled (with the plant or control number) microfuge tubes and store the extracts on ice for the GUS enzyme activity assay.
Assaying the Transgenic Plants (HOME)
Spectrophotometric Assay for GUS activity
Calculations
GUS Activity Assay by Histochemistry
Structural Evidence for Transgenic Plants
