Unit 3 extras

EXERCISE I
EXERCISE II
EXERCISE III

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.

Weigh out 0.7g of agarose and place the agarose into a 250mL conical flask.
Add 50mL of 1X TAE buffer, swirl to mix.
Notify your instructor that you are ready to microwave your agarose. Microwave in 20 second intervals to melt the agarose – a total of 60 sec will probably be sufficient.
The agarose solution can boil over very easily so keep checking it. It is good to stop it after each 20 seconds and give it a swirl. It can become superheated and NOT boil until you take it out - whereupon it boils out all over you hands. Hold the flask at arms length as you carry it back to your lab bench.
Leave it to cool on the bench for 5 minutes.
Check that your tray is positioned correctly in the chamber and sealed tight.
Pour the liquid gel slowly into the plastic container and push any bubbles away to the side using a disposable tip. The benefit of pouring slowly is that most bubbles stay up in the flask. Rinse out the flask immediately.
Insert a clean dry comb about 2 cm from the end of the gel and double check that it is correctly positioned (be sure the comb is clean). This is considered the top of your gel.
Normally, this new gel would be allowed to sit for a minimum of 30m. However, to save time, we will use gels created earlier today (like an instant cake on a cooking show!) for part 2.

Part 2: Next, we need to load and run our gel.

Pour 1x TAE buffer into the gel tank to submerge the gel to 2–5mm depth. This is the running buffer. You must use the same buffer at this stage as you used to make the gel.
Familiarize yourself with the samples you will be loading and into which lane they should be deposited . You will use 10 lanes in total, one for the ladder or marker, two for your positive and negative controls and the 7 samples.
Use a micropipette to load 10 µl of each sample into a well. Your instructor will demo this!
Close the gel tank. Remember that DNA has a negative charge and will migrate toward the positive (red) electrode. Assure the electrodes are connected properly to the power supply. Red is positive, and black is negative. Switch on the power-source.
Be sure the voltage (100V) and AMPs are set correctly. Ask your instructor to check the power supply, settings and hook-up.
Push “run.”
Check that a current is flowing. Look at the electrodes and check that they are evolving gas (ie. bubbles). If not, check the connections and assure the power-source is plugged in.
Monitor the progress of the gel by reference to the marker dyes.
Normally, this gel would run for 30-45 minutes. Instead, like on a baking show, the results of the gels have been recorded and made available to you in part 3.

Basic gel creation process...it's like jello!

Gel chamber and electrodes pictured with combs to create loading wells.

What are our controls? Click to enlarge.

What gets loaded where? Click to enlarge.

Diagram of electrical field and direction of migration. Click to enlarge.

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?

Collaborators

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.

View the Gel Results

1) Do you know enough about gel electrophoresis?

Following PCR, it is imperative to visualize your DNA. Last week we calculated how much we "should" have. Now we need to ensure we still have DNA and that it is the right gene. We need to ensure we have copied the cytochrome b gene. To do this, we will use a technique called gel electrophoresis.

First, review PCR from Lab 10.

Please review the first 4m 40s of this video from Khan Academy about gel electrophoresis.

Agarose gel electrophoresis is a method used in molecular biology to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. DNA-based gel electrophoresis can be used for the separation of DNA fragments of 50 base pairs up to several mega-bases (millions of bases).

After injecting your samples into the top of the gel, an electric field is used to push the charged DNA molecules through. The negatively charged phosphate groups of the sugar-phosphate backbone of DNA will migrate in an electric field away from the negative side (top) and toward the positive electrode (bottom).

Shorter DNA molecules move faster and migrate further down the gel.
Longer ones migrate slower remain closer to the top of the gel.

After allowing the sample to "run" for a specified time period, you can use various combinations of dye and light to visualize where your DNA stopped. The DNA will form a distinct band in the gel upon stopping. By comparing the distance traveled by each sample to fragments of known size (located in the ladder or marker, injected into the first lane of the gel), it's possible to determine the size of the fragments in each sample.

Exercise III. Determine PCR Yield

Once we know we have enough of the cytochrome b gene, we are ready for PCR.

Procedure: How much useful DNA will we have following PCR?

1) View this 3-minute video on PCR. You need to know the ingredients for PCR and why each is used. You also need to know the three steps and what happens in each one. You do not need to know the timing of the steps nor their corresponding temperature.

Research library

2) Visit our research library to learn a bit more about PCR. You need to know the ingredients for PCR and why each is used. You also need to know the three steps and what happens in each one. You do not need to know the timing of the steps nor their corresponding temperature.

3) We use an enzyme in PCR called Taq Polymerase. It mirrors the same action of DNA polymerase in our own cells during DNA synthesis.

3) Now, let's calculate our PCR yield: how many copies of the cytochrome b gene we may have in each sample following PCR (if all went according to plan). We performed 35 cycles of PCR on our bushmeat samples. Initial denaturation at 94 °C for 4 min and with a final extension at 72 °C for 10 minutes. The equation to calculate the final number of DNA strands created by PCR = N2^n, where N = the original number of DNA molecules to be copied and n = the # of PCR cycles.

4) Add these calculations for each sample to your spreadsheet.

2) Do you know enough about DNA bar coding?

DNA "barcoding" is like using an organism's DNA to establish its identity, similar to the bar codes on grocery items. But instead of using lines of vary widths, we use the sequence of nucleotide base pairs (As, Cs, Ts and Gs) in specific genes. Which genes to use depends on your taxa and your research question.

Review the slide presentation for a refesher on DNA structure.

Plant barcoding studies use one or a few plastid regions and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA.

Animal barcoding studies use a region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”) or the cytochrome b gene (CTYb).

Exercise II. What is the species of origin?

Once we've confirmed amplification via PCR through the gel electrophoresis, the samples are sequenced using a sequencing machine, which provides a read-out of the sequences of nucleotides within the sample. This process can be done in the WKU Biotechnology Center, or the samples can be shipped out for analysis.

Procedure: Confirm the actual species of origin for our samples and compare it to the putative species (what is was sold as).

1. View the resulting sequences for each of our samples HERE.

Sequence results show the order of nucleotides in our gene of interest for each sample. The order indicates species.

2. You are assigned to determine the species corresponding to your group number in lab. View the putative species for each sample HERE.

3. Visit the National Center for Biotechnology Information (NCBI) database called GenBank.

4. Follow the steps in the slide show below to match your sequence to all those stored in GenBank. Start at THIS LINK: The Genebank BLAST Page.

5. Once you've identified the samples' species of origin, add it to your data table along with the putative species.

This is a sequencer. PCR products are placed inside and the sequence of As, Tc, Cs and Gs is determined and the output is generated on the monitor.

Exercise II. What is the species of origin?

Procedure: Confirm the actual species of origin for our samples and compare it to the putative species (what is was sold as).

1. View the resulting sequences for each of our samples HERE.

Sequence results show the order of nucleotides in our gene of interest for each sample. The order indicates species.

This is a sequencer. PCR products are placed inside and the sequence of As, Tc, Cs and Gs is determined and the output is generated on the monitor.

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?

Exercise I. Is it the right DNA? Is it cytochrome b?

Exercise III. Class Discussion

Collaborators

1) Do you know enough about gel electrophoresis?

​Exercise III. Determine PCR Yield

2) Do you know enough about DNA bar coding?

Exercise II. What is the species of origin?

Exercise II. What is the species of origin?

Exercise III. Class Discussion

Collaborators

Exercise III. Determine PCR Yield