Lab 11: Pre-Lab

PLEASE READ! 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.  Due to the new "online only" nature of  some of these labs, you will be provided the output of various steps so you can complete the process of species identification without doing all of bench work. 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 Lab 10 and complete the corresponding LABridge in Blackboard.

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

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PCR vs. Intracellular DNA Synthesis

PCR is an artificial process completed inside a thermocycler, inside a lab. But! The process closely mirrors how our own cells replicate DNA during the S phase (synthesis phase) of the cell cycle: DNA synthesis.

Please review the information below to compare and contrast PCR (done in the lab) with what happens inside our cells, during S Phase, as DNA is replicated naturally. Review the YouTube vidoes below on PCR and DNA synthesis. Review the diagrams of each process and the enzymes involved in each.

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​Be sure you  know the ingredients (components) required for PCR to work. You also need to know what each one is and why it is required, AND the steps of PCR. We went over these in the post-lab for Lab 10. Check our library for a review as well.

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The process of polymerase chain reaction. Click to enlarge.

Taq-Polymerase operates in the same way in PCR. It is an enzyme from heat resistant bacteria and can function at the high temperatures used in the thermocycler (think back to CH 8 in 120).

The process of DNA synthesis is explored in CH 15 of your e-text.

DNA Polymerase is the enzyme primarily responsible for carrying out the process of DNA synthesis. There are several different types of DNA polymerase which have different functions throughout.

Gel Electrophoresis is our Next Step

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. 

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 unknow fragments are via comparision.
• See the example in the side bar.
• In this gel, there is just one fragment in each sample. We can tell the sample in lan 2 barely amplified enough in PCR. There is just a light band at about 650bp. Sample three's segment is about 600bp, 4 is about 450 bp, and the sample in lane 5 is about 400 bp long.​

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.

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

​Digestion liquefies the tissue in such a way that keeps the DNA intact for extraction. We use special "kits" as pictured to streamline the process. Extraction requires more steps to break through the cell and organelle membranes to free the DNA. 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 your own cheek cells.

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 10, 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.

​If you feel confident with this material, click the bridge icon and navigate to Blackboard to take the LABridge. Be sure you have saved and completed the Lab Notebook Guide from Lab 10.

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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 visualize and measure our PCR product via gel electrophoresis to ensure we have copied the correct gene for bushmeat identification to species. 

Exercise I. Prepping for Gels
Exercise II. Gel Electrophoresis
Exercise III. Determine PCR Yield

Lab 10 - 12 Objectives (click to enlarge).

• Exercise I
• Exercise II
• Exercise III

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Exercise I. Making the Agarose Gel

Did the PCR work? Did we copy the CYTb gene?
​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: 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. Check that your tray is positioned correctly in the chamber and sealed tight. 
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 Exercise II.

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

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

Exercsie 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 dark bands.

Procedure

1. 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.
2. 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. 
3. If you still have questions about the controls, read over the info 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 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 Exercise III.

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

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

What gets loaded where? Click to enlarge.

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

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.

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 if there 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 in a transilluminator. he compound forms fluorescent complexes with nucleic acids which can be viewed under UV light. 
3. You have been provided with a photo of the gel and it's marker so you can read the results (in the sidebar).
4. 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.
5. Record these results in your spreadsheet from Lab 10.
6. 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.​
7. 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.
8. 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!
9. 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)?
10. 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.
11. 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.
12. 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. ​
13. 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 8: Enzymes
​Chapter 12: The Cell Cycle
Chapter 15: DNA and the Gene: Synthesis & Repair
Chapter 16: How Genes Work
Chapter 20: The Molecular Revolution
Chapter 54: Biodiversity and Conservation Biology *BIOL 122