Lab 11: Pre-Lab

Over the next several years BIOL 121 students will be testing meat samples from Kenya, through DNA analysis, to determine if poaching and bushmeat use is threatening conservation efforts.  We are currently testing the procedures that will be used for this research project. You will be provided the output of various steps so you can complete the process of species identification. In labs 10-12, your task will be to identify the species of origin of a meat samples from Kenyan butcheries. You will learn about poaching, the bushmeat crisis and practice key techniques to complete DNA analysis of your samples. To prepare for Lab 11, please review this pre-lab. Once you feel confident regarding the below topics, and have your Lab Notebook ready from last week, complete the corresponding LABridge in Blackboard.

• Introduction/Review
• Do you know enough?
• What will we do in lab?
• LABridge

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Review the steps of DNA Analysis

In Lab 11 we will run a gel to make sure we amplified DNA and that we made copies of the correct gene. We will also try to determine how much DNA we have to complete our analysis following amplification via PCR.

Be certain you're comfortable with PCR! Most of your LABridge questions this week will be on step three! It is a tough topic so extra review is warranted!

Please review the steps and sequence below, with a focus on steps 3 & 4.

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1) Sample PROCESSING

Bushmeat Processing: Using aseptic techniques, the bushmeat samples are cut into approximately 1 cc sections. They labeled and stored in ethanol at -20 degrees Celsius. Special care is taken to ensure no cross-contamination or human contamination occurs. Samples are then carried back or shipped to the WKU biotechnology Center. -----> This has already been done.

2) Digestion & Extraction

This process is similar to the extraction we completed with the strawberry. However it involves many more steps and results in a cleaner product with far less protein. We use special "kits" as pictured to streamline the process. Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. Once extraction is complete, the DNA sample is tested to ensure an adequate amount of intact DNA was extracted from the sample. ​-----> This process has already been done with our meat samples. In Lab 10, you will practice extracting DNA from a strawberry to better understand this process

3) Polymerase chain reaction (PCR)

PCR makes copies of a DNA fragment from one original copy. The goal is to amplify a specific region, the target DNA or gene of interest (GoI), depending on the type or goal of research. The PCR cocktail includes the following ingredients:  the DNA sample, primers (short sequences of RNA or DNA that start replication), dNTPs  (free nucleotides), taq polymerase (a heat stable form of DNA polymerase derived from bacteria) and a buffer solution. There are three steps to PCR in which the temperature is cycled (in the thermo"cyler"). You need to know the steps and what happens in each! The total number of resulting DNA strands is (the number of original strands) X 2^n, where n = the number of PCR cycles. -----> In Lab 11, you will be given PCR products from our meat samples in lab and asked make some calculations.

4) Gel electrophoresis

Agarose gel electrophoresis is a method used to separate DNA strands by size, and to determine the size of the separated strands by comparison to strands of known length. ​Your PCR products are deposited in the top of the gel. Using electricity, the DNA (with a negative charge) is pushed through the gel towards the positive electrode. As your gel "runs," the DNA is separated by size. The DNA strands show up as bands under UV light and you can read the results. Your products can be compared with the ladder or marker, which has standard sized DNA fragments of KNOWN length used for comparison. In this way, you can know the exact length of your DNA samples. -----> You will MAKE & RUN an agorose gel in Lab 11 to make sure our PCR product contains the cytochrome b gene.

5) Sequencing

Once we know we have amplified (copied) the right gene we are ready to sequence the gene. We expect the sequence (the order of As, Ts, Cs and Gs) within the cytochrome b gene to be different for different species. Samples are placed into a sequencer apparatus which can detect the order of nucleotide bases in our sample. The sequence is then cleaned and edited,

6) BLASTing

6) BLASTing: The National Institute of Health (NIH) and National Center for Biotechnology Information (NCBI) hosts a database called GenBank, which houses all known DNA sequences. Once the sequences of our samples are ready, they are pasted into a search tool (called a BLAST) which matches them to the correct species! -----> You will be provided the sequence of successful samples in Lab 11 and asked to determine the species of origin in lab.

​Review the comparison of PCR with DNA synthesis. 

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You'll review the entire process of DNA synthesis in BIOL 120. In it, DNA polymerase builds the new copies of DNA strands from the original template.

We use Taq-Polymerase in the same way in PCR. It is an enzyme from heat resistant bacteria (Thermus aquaticus) and can therefore function at th ehigh temperatures used in th ethermocycler.

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 (the right length). We need to ensure we have copied the cytochrome b gene. To do this, we will use a technique called 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 PCR samples (plus a special dye) 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). 
• 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. These are called "bands."
• 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.

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Please review the first 4m and 40s of this video from Khan Academy about gel electrophoresis. Review the bullet points below and the example image in the sidebar. 

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​Review the gel example and accompanying explanation below.

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• Shorter DNA molecules move faster and migrate further down the gel.
• Longer ones migrate slower remain closer to the top of the gel.
• You can tell how long each fragment is by comparing them to the ladder, usually placed in the first and last lanes.
• The ladder contains fragments of known size so you judge how long your unknown fragments are via comparison.

See the example in the side bar. In this gel, there is just one fragment in each sample.

1. Lane 1: DNA ladder/ marker
   
2. Lane 2: The sample did not amplify enough in PCR.
3. Lane 3: One fragment of ~650bp. The band at the very top is from the well itself.
4. Lane 4: One fragment of ~450 bp.
5. Lane 5: One fragment of ~400 bp.
6. Lane 6: Another DNA ladder/ marker
   

What will we do in lab & how will we do it?

During Lab 10, we studied DNA extraction and practiced on different types of fruit. We received the extraction results of our bushmeat samples and calculated the potential amount of mtDNA and of CYTb (our "GOI" gene of interest) we could have in our samples. Lastly you completed a virtual lab on PCR. For the purposes of Lab 11, let's assume the PCR has been run on the extracted products of our bushmeat samples. In this lab, we will continue with Gel Electrophoresis.

Lab will precede in three parts.

1) Make your gel: You will make a "gel" in a process similar to making Jello. 

2) Conduct Gel electrophoresis (i.e., run your gel) : You will place a small amount of the PCR product from each bushmeat sample into the gel. If we synthesized the correct DNA, all our samples should be about 490 base pairs long.

3) Results: You will view these results to determine if our PCR was successful.
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Click here to get to WKU's blackboard to take your LABridge for this week. Be sure your Notebook Entry from last lab is ready to submit!

Lab 11: Protocol

Your task in Lab 11 is to identify the species of origin of a meat sample from a Kenyan butchery.

Exercise I:  Make your gel

Exercise II: Conduct Gel electrophoresis (i.e., run your gel)

Exercise III: Results: Review the results.

Lab 10 - 12 Objectives (click to enlarge).

• Exercise I
• Exercise II
• Exercise III

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​Exercise I. Making the Agarose Gel

​So, did our PCR work? Were we able to amplify the DNA as intended? At this point in the process, we have no idea!!!! In fact we might not have ANY DNA. 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!

Materials.

Digital Balance

Agarose Powder

TAE Buffer

Flask for Gel Mix

Microwave

Casting Tray

Procedure: First we need to create our gel.

1. Weigh out 0.7g of agarose and place the agarose into a 250mL conical flask.
2. Add 50mL of 1X TAE buffer, swirl to mix.
3. 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.​
4. 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.
5. Leave it to cool on the bench for 5 minutes.
6. We will be using steel trays to form our gels. It is vital that you wet down the rubber inserts on each side before attempting to pace your mold into the tray. 
7. 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.
8. 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.
9. 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.

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

Casting tray and plastic mold used for setting gel. Click to enlarge.

Exercise II. Loading & Running the Gel

We will conduct agarose gel electrophoresis 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 bands.

Materials.

Pre-Made Gel

TAE Buffer

DNA Samples

Pipette & Tips

Power Supply

Gel Chamber

Procedure. Run your gel.

1. Your "pre-made" gel will be in the gel rig or you may need to remove it from the metal tray. Follow your TA's instructions and BE CAREFUL!
2. Once your gel is positioned in the rig, 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.
3. 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. Review controls below.
4. Use a micropipette to load 10 µl of each sample into a well. Your instructor will demo this!
5. 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. 
6. Be sure the voltage (100V) and AMPs are set correctly. Ask your instructor to check the power supply, settings and hook-up.
7. Push “run.”
8. 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. 
9. Monitor the progress of the gel by reference to the marker dyes.
10. 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.

The red and black electrodes are plugged into the power supply and the current pushes your samples down the gel.

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

What gets loaded where? Click to enlarge.

What is looks like to load a gel.

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

We use two types of controls in DNA analysis. We start running both from the very beginning, through PCR, onto to gel electrophoresis and through to sequencing. If either control results in an incorrect identification, we have to start the ENTIRE process over and all our results are considered invalid

A positive control

• Is a known DNA sample with target DNA.
• It should always work well in your PCR and therefore, should always show up as expected in your gel (at the right length).
• It should always show up as the right sequence for identification.
• In this experiment we used impala DNA as our positive control or C+.
• At the end of all of our steps of DNA analysis, we should determine the species ID of this C+ to always be impala.
• This would be a positive result for bushmeat. 
• This tests the success and validity of our protocol.
• If this C+ shows up to be ANYTHING BUT impala, all our results are invalid and we would need to start all over again with fresh extractions.

A negative control

• Is a known sample with NO DNA.
• Following PCR is should NOT show up on your gel at all because there should be no DNA for amplification during PCR.
•  It should never produce a sequence for identification.
• In this experiment (as in most) we used distilled water for our negative control or C-.
• At the end of all of our steps of DNA analysis, we should NEVER find any DNA from this sample.
• This tests for any contamination that may have occurred.
• For example, if we sequenced the C- and found human...we would have made a big mistake and would need start over again with fresh extractions.
• Or, for example, if we sequenced the C- and found cow or impala...we would have made another type of big mistake and would need start over again with fresh extractions.

What are our controls? Click to enlarge.

​Exercise III. View & Analyze the Results

1. After electrophoresis is complete, a light band of loading dye appears across the bottom, however what you CANNOT see are any bands of DNA.
2. 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.
3. View the RESULTS!​​
4. Were all of our samples amplified in PCR?
5. Did we amplify the right gene? Remember we need a 490bp region of the cytochrome b gene. Review why.
6. Record these results in your spreadsheet from Lab 10.
7. Following sample processing and DNA extraction you made some calculations regarding how much CYTb DNA we might have.  Now that we know we do have some DNA, we need to determine how CYTb DNA we may have following PCR.​
8. First, you are dealing with both very small and very large numbers in these calculations. If you need a refresher on scientific notation, try this resource.
9. First, retrieve and open your excel data sheet with your calculations from Lab 10. If you need a new copy it is in the sidebar, but I hope you saved your calculations from Lab 10!
10. Now, let's calculate our PCR yield: how many copies of the cytochrome b gene might we may have in each sample following PCR (if all went according to plan)?
11. Please note: We performed 35 cycles of PCR on our bushmeat samples.  Initial denaturation at 94 °C for 4 min with a final extension at 72 °C for 10 minutes.
12. The equation to calculate the final number of DNA strands created by PCR = N(2^n), where N = the original number of DNA molecules to be copied (which you calculated in our last lab) and n = the # of PCR cycles.
13. Use this equation to determine how many CYTb copies we have following PCR for each of our samples, and add these calculations for each sample to your spreadsheet. ​
14. Download and complete the Lab Notebook Guide for Lab 11. Be sure everyone saves a copy for next week's LABridge.

View the Gel Results

Example gels. Click to enlarge.

At the end of this first cycle, there are two DNA copies instead of the one original copy. The process continues, doubling the number of target DNA copies with each cycle. 
The general formula for the number of DNA strands created by PCR is
N(2^n) 
N = the number of original strands
n = the # of PCR cycles.

Lab 11 BIOL 120 CONNECTIONS
Section 1.6: Doing Biology
Big Picture 1: How to Think Like a Scientist
Chapter 4: Nucleic Acids
Chapter 15: DNA and the Gene
Chapter 20: The Molecular Revolution
Chapter 54: Biodiversity and Conservation Biology *BIOL 122