Lab 9
Reporter Genes
GUS
The GUS gene is used in transgenic studies to verify the transmission of the
transformation vector into the target plant. It is informative because nothing in the plant
tissue would stain blue; the blue stain can only mean the plant is transformed. GUS
makes a protein that turns blue when mixed with X-gluc (5-Brom-4-chlor-3-indolyl-ßDglucuronide). Chimeras can be observed in the X-gluc stained tissue.
X-gluc stain (prepared):
2.5ml
0.2M Na2HPO4
2.4ml
100µl
D.I. water
0.5M Na2EDTA
5.0mg X-gluc
5ml
1. Put 30-40µL fresh X-gluc stain into the well of a microtiter plate.
2. Select a shoot from your Tobacco Transformation Experiment. Cut a thin section
of leaf or stem and place it into an individual well - keep track of where sections
come from.
3. Stain sections for 4-5 hr. at 37 in the incubator.
4. After staining, clear or fix the tissue by adding 50µL of 95% ethanol:glacial acetic
acid (3:1 v/v). Wait a half hour before scoring for GUS. Perform step under
Hood.
5. Under the dissecting microscope, score the tissues for GUS (blue) staining.
6. Note fully GUS and chimera tissues. Glacial acetic acid can damage
microscopes - leave the cover on the microtiter plate.
GFP
GFP, or green fluorescence protein, is becoming the reporter gene of choice for
transgenic studies. As the name implies, the GFP gene product will fluoresce green
under UV light. Using GFP allows plants to be scored without damaging the tissue.
Entire plant organs can be viewed under the dissecting scope equipped with fluorescence
capability.
For this lab section, use the fluorescence dissecting scope to view your transgenic
tobacco and Arabidopsis.
Total DNA Extraction
Procedure:
DNA extraction using DNAzol ES (MRC, Inc.)
1. Weigh out 250mg of Tobacco leaf tissue and place in a mortar.
2. Pour in liquid Nitrogen.
3. Grind tissue with a pestle and mortor until tissue is a powder.
4. Add 750l ml of DNAzol ES.
5. Mix well and incubate for 5min at room temperature.
6. Add an equal amount chloroform (1:1) to tubes.
7. Centrifuge at 12,000g for 10min.
8. Transfer supernatant to a clean tube
9. Add an equal amount chloroform (1:1) to tubes.
10. Centrifuge at 12,000g for 10min.
11. Transfer supernatant to a clean tube, add a 525l of 100% ethanol.
12. Invert the tube to mix and incubate for 5min at room temperature.
13. Centrifuge for 4min at 5000g.
14. Discard Supernatant.
15. Prepare DNAzol ES-EtOH wash by mixing 1 volume DNAzol ES with 0.75 volume
100% EtOH. (this has been prepared for you)
16. Resuspend pellet in 150l EDTA. Incubate for 10-20 min at 50C
17. Add 1.5ml DNAzol ES-EtOH wash. Incubate 5 min. at room temperature.
18. Centrifuge for 4min. at 5000rpm.
19. Discard supernatant, add 1.5ml 75% Ethanol. Vortex intermittently for 5min.
20. Centrifuge for 4min at 5000rpm.
21. Discard supernatant and pipette to remove excess ethanol.
22. Dissolve pellet in 35l sterile DDI water
23. Transfer to 1.5ml microcentrifuge tube, label, store at -20C.
PCR
In a 0.5ml microcentrifuge tube add the following in order:
50ul reaction
22.75ul
sterile H2O
10ul
5X Taq buffer (Mg free, Promega)
1ul
dNTP (5mM, Promega)
5ul
MgCl2 (Promega)
0.5ul
Primer1 (100uM) ** be sure to use the correct primers!!**
0.5ul
Primer2 (100uM)
10ul
DNA template
0.25ul
go-Taq Polymerase (Promega)
PCR reaction conditions in MJ thermocycler:
1 cycle:
94C 2min
30 cycles:
94C 30sec
55C 30sec
72C 45sec
1cycle:
72C 2min
hold at 4C