​ Figure 5. Schematic concept of polymerase chain reaction.

​ Figure 6. Schematic concept of gel electrophoresis.​

• Upon completion of this exercise students will be able to
• Differentiate between genes and alleles in terms of genetic loci and observed traits.
• Diagram the process of PCR and explain its value in molecular biology.
• Extract DNA from plant tissues and analyze its genotype using PCR (Fig. 5).
• Predict the phenotype of plants given gel electrophoresis data from the Kap PCR products (Fig. 6).

Planting OWB Seeds

Materials

Pots (13 cm [5 in.])

Seed Packet
Plant Tag
Masking Tape
Fine-Point Permanent Marker
Standard Potting Mix

Planting Instructions

1. Obtain a pot, marker, tag, tape, and seed packet from instructor.
2. Label your tag with the OWB number (from the seed packet label), date, class period, and your name. Label the tape the same way and apply it around the top of the pot. This will be used to identify your plant from others.
3. Fill the pot to the top with soil in a scooping motion, but do not compact the soil into the container.
4. Place your finger into the soil to the first knuckle (~2.5-cm [1-in.] deep) three times to make three separate spaces for the seeds.
5. Drop one seed in each of the three holes that you created, lightly cover with remaining soil.
6. Place the tag into the soil for easier identification. Place the pot under the light bank (or growing area).
7. Water your plant so the soil is moist. Make sure the seed does not float to the top.
8. Seedlings should emerge within 1 week.

Leaf Tissue DNA Extraction

Materials

• Microcentrifuge Tubes (2.0 ml) and Markers
• Leaf Tissue (0.3 g)
• Tube and Pestel or Glass Rod
• Glass Slide
• Tube Holder
• Pipettes and Tips
• Razor Blade
• Vortex
• Centrifuge
• Fume Hood
• Freezer
  
• Gloves
• Water Bath
• Ice

Reagents and Buffers

2´ CTAB Buffer
20% (w/v) Sodium Dodecyl Sulfate (SDS)
5 M Potassium Acetate (Stored at –20°C)
Absolute Isopropanol (Stored at –20°C)
70% Ethanol (Stored at –20°C)
TE Buffer
2-Mercaptoethanol**
Rubbing Alcohol
** 2-Mercaptoethanol is toxic and dangerous for the environment. Therefore, it should not be thrown away in the trash. The tubes should be left open under a fume hood to allow the chemical to evaporate from the leaf tissue. Once evaporated, the tubes are safe to throw away in the trash.

The odor of 2-mercaptoethanol is like the odorant added to natural gas. Vapors can irritate the eyes and mucous membranes. The amount being used in this extraction is minimal, and the instructor should be the only person to handle the stock solution in a fume hood.

Day 1: Harvest Plant Tissue

1. Label the top and side of a 2.0-ml centrifuge tube with the plant number, class period, date, and name, using a fine-tip permanent marker.
2. Clean the glass slide, razor, and glass rod with rubbing alcohol to remove any foreign DNA.
3. Collect 3–4 leaves at about 7 days after planting (they should be 7–10 cm [3-4 in.] long). Find the mass of the leaves until you obtain ~0.3 g of leaf tissue.
4. Place leaves on the clean glass slide. Chop the tissue into very small pieces using a clean razor blade. The more finely chopped the better DNA extraction will be.
5. Immediately transfer tissue to the labeled 2.0-ml microcentrifuge tube and further grind the tissue with the clean glass rod. Mash the tissue into a wet pulp for 2 min. (label with the plant line, date, and initials of the student).
6. Add 800 µl of CTAB buffer and 2-mercaptoethanol solution from the tube labeled CTAB-2Mercap and 100 µl of SDS from the tube labeled SDS. Shake by hand or vortex gently to mix.
7. Place your microcentrifuge tube on ice and give to the instructor for overnight storage.
8. Clean Up: Clean your slide, razor, and glass rod with rubbing alcohol. Make sure each tube is properly labeled as the students give them to the instructor. The permanent marker can often get rubbed off. Store these samples overnight at 4°C or freeze for longer storage.

Day Two: Extract DNA

1. Allow your sample to thaw if necessary. You can speed this up by holding the microcentrifuge tube in your hand. Use the vortex to mix up the contents by holding the microcentrifuge tube down on the rubber disc for 15 sec, or until all the material is mixed.
2. Incubate the tube at 65°C for 10 min. Place the tube in the tube holder in the water. Make sure not to disrupt other tubes.
3. While you are waiting, put crushed ice in your cup for the next step and get a tube of cold potassium acetate and tube of cold absolute isopropanol (labeled AI) from the instructor and put them on the ice.
4. After 10 min has elapsed, remove your tube from the water bath and place it on your ice.
5. Add 410 µl of cold potassium acetate. Mix by inverting the tube up and down 10 times. Place the tube back on ice for 3 min.
6. Keep your tube on ice until all groups are ready. Then, bring your tube to the instructor to place in the centrifuge. The tubes will spin at 13,200 rpm for 22 min at room temperature. While this is taking place, obtain a new 2.0-ml microcentrifuge tube and label it with the plant number, class period, date, and name (same as the first tube).
7. When the centrifuge has stopped, obtain your tube, and return to your lab bench. Transfer approximately 1 ml of the supernatant to the new 2.0-ml microcentrifuge tube. This can be tricky as you must place the pipette tip past the film on top of the liquid and only draw up the liquid above the green plant matter (which should be clumped together at the bottom of the tube). There should not be any green material drawn up into the pipette tip.
8. To ensure you get ~1 ml of clear supernatant, you may need to repeat the centrifugation step with the same the tube.
9. Add 540 µl of ice-cold absolute isopropanol. Invert the tube 10 times to mix, place the tube back on ice for 20 min, and bring to the instructor for overnight storage at 4°C or in a –20°C freezer for longer storage. Bring your used tube with plant tissue in it to the instructor for disposal (DO NOT THROW AWAY OR PUT DOWN DRAIN).

​Day Three: Purification of DNA

1. Allow your sample to thaw if necessary. You can speed this up by holding the microcentrifuge tube in your hand. Use the vortex to mix the contents by holding the microcentrifuge tube down on the rubber disc for 15 sec, or until all of the material is mixed.
2. Centrifuge at 10,200 rpm for 10 min. Your tube should now contain a clear solution and a small pellet of DNA that is stuck to the wall of the tube just above the bottom. Discard the supernatant by pipetting it out. Be careful not to disturb the pellet.
3. While you are waiting, put crushed ice in your cup and get a tube of 70% ethanol (labeled E) and a tube of TE buffer (labeled TE) from the instructor and put them on the ice.
4. Wash the pellet once with 500 µl of 70% ethanol. Gently invert the tube several times; do not break up the pellet. If the pellet dislodges from side of tube or breaks apart, centrifuge the tube again at 10,200 rpm for 5 min. Pipet the excess ethanol from the tube, again being careful to avoid disturbing the DNA pellet. Let any excess drops on the side of the tube dry.
5. Add 200 µl of TE. Vortex gently to resuspend the DNA pellet in the TE buffer. Give the tube to your instructor for overnight storage.
   

Day Four: PCR of the Kap Gene

Materials

• Thermal Cycler
• Centrifuge Tubes (1.5 ml)
• Cup, Ice
• Micropipettes
• Pipette Tips
• Kap PCR Primers​
  
• Molecular-Grade Water
• DNA Template(s)
• PCR Tubes (0.2-ml)
• DNA Taq Polymerase (Beads or Master Mix)

Instructions for PCR

1. Obtain your DNA in the 2.0-ml microcentrifuge tube. Begin to thaw it. Obtain a PCR tube with a Taq polymerase bead at the bottom and label it like the other tubes with your OWB number, class period, date, and initials (you can just use your initials instead of your full name since the tube is small).
2. While you are waiting, put crushed ice in your cup.
3. Make sure the bead is at the bottom of the tube. Your instructor will add 24 µl of the hooded/awn (Kap) primer mix to your PCR tubes.
4. Add 1 µl of your DNA template to your PCR tube.
5. Vortex the tube until the bead fully dissolves and the solution is clear.
6. Tap the PCR tube on the table to get all the solution down to the bottom of the tube.
7. Store tubes on ice until instructed to transfer your tubes to the thermal cycler.
8. Give your tube of DNA back to the instructor for storage at –20°C.

Day Five: Using Gel Electrophoresis to Analyze PCR Products

Materials

• 1´ TBE Buffer
• Flask (500 ml)
• Wax Paper or Parafilm
• Gel Box, Transilluminator
• Agarose
• Gel-Loading Dye
• Lab Tape
• Balance or Scale
• Micropipettor and Tips
• Microwave
  
• Hot Glove
• GelGreen DNA Stain

Preparing and Loading the DNA

1. Obtain you tubes containing PCR products from the instructor.
2. On a piece of wax paper or parafilm, combine 3 µl of loading dye with 10 µl of PCR product. Draw up and dispense the mixture with your pipette three to four times to mix it. Your product should be a blue color once mixed.
3. Take your wax paper over to the gel electrophoresis. Add 10 µl of the blue DNA/dye mixture to the correct gel well indicated by the paper and your instructor.
4. Repeat steps 1–3 for each sample you want to load onto the electrophoresis gel. If you are going to be loading numerous samples, it may be helpful to draw a grid on your wax paper to indicate which DNA samples are which.
5. Be sure to use a new tip for each sample! Don't worry about getting the micropipette tip point down into the gel well. Hold the tip over the gel well you are targeting and dispense the DNA. The loading dye causes it to sink down into the well.
   

When all of the samples have been loaded and recorded, place the lid on the gel box. Plug the leads connected to the lid into the power source and turn on the current. Run the gel for 30 min at 100 V. (You may have to run it longer than 30 min.)

​ Figure 7. Basic structural diagram of DNA organization and composition.​

​​ Figure 8. GelGreen staining of Kap amplicons from Oregon Wolfe barley parents and doubled haploid progeny, as visualized on the transilluminator.​

How Gel Electrophoresis Works

Looking at a strand of DNA (Fig. 7), we can see that it looks like a ladder, with the “rungs" being the bases (A, T, C, or G) and the sides of the ladder consisting of sugar molecules joined together with phosphate molecules. The phosphate molecules give the entire DNA molecule a net negative charge. The DNA molecule is normally a double helix, resembling a spiral staircase.

Because DNA is negatively charged, it will migrate from a negatively charged anode (black) to a positively charged cathode (red) in an electric current. You can use the mnemonic “run to red" as you put the lid on the electrophoresis chamber. It is important to note, the larger the DNA fragment, the harder it is for the DNA to move around the agarose, so it moves more slowly. This means that given the same amount of time the shorter fragments will move further and the larger fragments will stay closer to the wells where you added your sample.

Visualizing the Gel

1. The DNA is not visible at this point, but during the electrophoresis the GelGreen stain is intercalated into the DNA. To see the DNA, gently remove the gel from the gel box or plastic bag/wrap and place it on the blue platform of the Vernier transilluminator. Lower the orange lid, and turn the light knob. The DNA bands should become visible (Fig. 8). The darker the surroundings, the better the bands show up. Turning off the room lights room can help.

Discussion Questions

1. ​How are genes and alleles related?
   
   Answer: Genes specify which trait; alleles specify what form the gene takes (e.g., alleles are different forms or variants of one gene; even a single nucleotide polymorphism (SNP) can alter the gene to make a new allele).
   
   
2. What analogy could you use to explain the difference between a gene and an allele?
   
   Answer: A gene provides the sequence that encodes the trait; alleles of that gene specify the form of the trait (e.g., for a gene for eye color, different alleles may specify green, blue, or brown).
   
   
3. We are purifying DNA from a small segment of the barley genome; how can we be sure this plant tissue has the genes we are investigating?
   
   Answer: Try isolating DNA from the leaves and amplifying the gene via PCR with specific primers.
   
   
4. Why do scientists use PCR?
   
   Answer: To amplify segments of DNA.
   
   
5. What determines how fast a segment of DNA will move through the gel?
   
   Answer: The size of the DNA fragment, the makeup of the electrophoresis buffer, and the amount of voltage or current applied.
   
   
6.  Would you expect the Kap allele to migrate faster or slower on a gel?
   
   Answer: Slower, because it is 305-bp larger.
   
   
7. Would this type of analysis (PCR and visualization) allow researchers to separate alleles that only differ in DNA sequence, not size? Why or why not?
   
   Answer: At face value, no. But, if you add an additional step, typically called cleaved amplified polymorphism (CAP), where the amplified DNA is cut with an enzyme that differentiates between the different alleles (i.e., one allele has a particular restriction site and the other allele does not), the resulting lengths of the fragments will be different, and you can visualize the size differences by electrophoresis.​

​​Overview​​Experiment 2​​Experiment 3
​iTAG Instructor’s Planning Resources
​​AppendixGlossary​​

​Acknowledgments​

We thank the Iowa State University Research Experience for Teachers (RET) for connecting teachers with career scientists to further their professional development and Dr. Pat Hayes of Oregon State University for OWB-ISS population resources. This program was funded, in part, by National Science Foundation–Plant Genome Research Program Grant 13-39348; USDA-Agricultural Research Service Projects 3625-21000-067-00D and 5030-21220-068-000-D to RPW; USDA-National Institute of Food and Agriculture Grant 2020-67013-31184 to RPW, NB, and NP; and Iowa Agriculture and Home Economics Experiment Station (IAHEES) Project 4208 to NP and NB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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