Exercise I. Is it the right DNA? Is it cytochrome b?
We have no idea!!!! In fact we might not have ANY. The calculations you completed in Exercise I are the best case scenario. The truth is PCR often fails. There are many reasons! Too much enzyme, low quality DNA, poor primers, low primer concentrations, incorrect PCR cycling, and so on! We need to SEE our DNA to know it exists and to confirm it is the right length!
Procedure: Conduct agorose gel electrohphoresis to confirm amplification of the right DNA. We need a 490 bp section of the cytochrome b gene. So all our samples should be approximately 490 bp in length and result in dark bands.
Part 1: First we need to create our gel.
Part 2: Next, we need to load and run our gel.
Part 3: Lastly, we need to interpret the results. What do they mean?
- After electrophoresis is complete, a light band of loading dye appears across the bottom, however what you CANNOT see are any bands of DNA.
- In order to visualize the DNA the gels must be stained with ethidium bromide (EtBr) and exposed to UV light. The compound forms fluorescent complexes with nucleic acids which can be viewed under UV light. You have been provided with a photo of the gel and it's marker so you can read the results.
- View the RESULTS!
- Were all of our samples amplified in PCR?
- Did we amplify the right gene? Remember we need a 490bp region of the cytochrome b gene. Review why.
- Record these results in your spreadsheet from Lab 10. If you did not save the spreadsheet from last week, here it is.
Exercise III. Class Discussion
We are going to discuss our results and this project as a class. Before we start here are some questions we might discuss:
- Why is it important to understand bushmeat use in Kenya?
- Is bushmeat use for private needs different than a commercial enterprise wherein poached meat is sold to the public? Why or why not?
- Could these types of practices happen here?
- Do you think they could have an impact on conservation initiatives or public attitudes toward wildlife?
- What factrs in Kenya are contributing to this crisis in Kenya?
- How can or should we move forward?
ACKNOWLEDGEMENTS: CO-AUTHORS. Science is a collaborative process. Simon Kisaini (at left) has been working to conserve Kenya's wildlife since his youth and continues to do so now through Wildlife Works in Kasigau. He designed our sampling and field preparation protocols. Naomi Rowland (at right) is the Lab Coordinator for the WKU Biotechnology Center and developed and tested the molecular protocols for this research.