Structural Evidence for Transgenic Plants
PCR Analysis of Transformed Plants
The goal of this procedure is to determine that your kanamycin-resistant plants are transformed by testing directly for the introduced DNA using the polymerase chain reaction (PCR) and gel electrophoresis.
The screening of transformed plants for the presence of an introduced gene used to be a time consuming process that involved the isolation of genomic DNA and Southern blot analysis. The polymerase chain reaction (PCR) however, has made this a relatively simple task that can be completed in an afternoon. In this lab you will use PCR to detect the GUS gene. The gene contains approximately 500 to 600 base pairs.
REDExtract-N-Amp™ Plant PCR kit (Sigma Product # XNAP http://www.sigmaaldrich.com)
Each team should test a control and all the experimental plants that you tested for GUS enzyme assay. Your instructor will amplify by PCR the gusA gene on the pBI121 plasmid to run on one of the gels as a positive control.
For each plant or control to be tested:
- Collect 1 disk of young leaf tissue from a transgenic plant .by punching it out with the top of a microfuge tube. Wear gloves so that you do not contaminate the tube or the leaf with anything that might contain DNA and DNAase (your hands).
- Push the leaf disk into the bottom of the microfuge tube using a tip from your P200. It is ok if the tissue tears, but be gentle.
- Add 100 µl of Extraction Solution to the microfuge tube containing the leaf disk. Make sure the disk is completely covered with solution. Close the tube and vortex briefly.
- Incubate for 10 minutes at 95 C. NOTE that only one side of the heat block heats to 95C. If you are using the dry bath note that the entire surface is HOT. If using a water bath, be careful not to burn your fingers when placing tubes into and taking tubes out of the water bath. Use a cap holder to make sure your tubes do not becoming uncapped during the incubation.
- Remove tube from the incubator. The leaf tissue usually doesn’t appear degraded after this treatment.
- Add 100µl of Dilution Solution, cap the tube, and vortex to mix.
- Place the diluted DNA extracts on ice. You do not need to remove the leaf disk.
NOTE: The exact ingredients for the solutions in the kit are proprietary, but the extraction solution probably contains NaOH and the dilution solution used to neutralize the NaOH is probably HCl in a Tris buffer with detergent.
PCR Amplification of gusA
- Change your gloves and use precautions to avoid contamination of the master mix cocktail with DNA from the extracts and between extracts. Your team has been given 6 pcr tubes/ group with 16µl of PCR cocktail in each thin-walled labeled PCR tube of the appropriate color for your team. Label the tubes with the plant number or control plant # information on the side AND on the top of the tube. The final tube is the negative control that will have no DNA added to it.
- Add 4 µl of the diluted leaf extract to the PCR cocktail. Wash the tip by pipetting up and down once to ensure all leaf extract is dispensed. Vortex briefly and gently to mix. Change tips and repeat for each extract, taking care not to cross contaminate samples.
- Someone in the class will be assigned to set up and run a negative PCR control by adding 4 µl of HPLC water to 16µl of PCR cocktail and running it in the thermal cycler with the rest of the reactions.
- Tap gently or shake down the PCR tubes to be sure all reagents are in the bottom of the tubes.
- With the help of your instructor place your reactions in the thermal cycler and run the following cycle: (3 min; 94°C; 1 cycle)(30s; 94°C : 30 s; 55°C : 1 min 72°C; 35 cycles)(10 min; 72°C; 1 cycle)
- After PCR your instructor will transfer the PCR products into the freezer until next week when you will digest the PCR product with restriction enzymes and run the DNA on an agarose gel.
PCR cocktail is REDExtract-N-Amp PCR Ready Mix containing: buffer, salts, dNTPs, Taq polymerase, RED Taq dye and jumpstart Taq antibody.
GUS Activity Assay by Histochemistry
Restriction Enzyme Digest of PCR product
Assaying the Transgenic Plants (HOME)
Leaf Extract Preparation
Spectrophotometric Assay for GUS activity