Laboratory Exercise: ISOLATION OF DNA FROM WHEAT GERM In this lab, you will: Isolate DNA from a eukaryotic source Run gel electrophoresis to visualize the DNA Optionally cut the genomic DNA using the restriction enzyme EcoR1 All cells contain nucleic acids. Bacterial cells have no nucleus and therefore have their DNA unbounded by a nuclear membrane. It is relatively simple to isolate DNA from a prokaryotic cell. This exercise will provide the opportunity to easily isolate DNA from a eukaryotic source. Using products purchased in a grocery store, you will lyse wheat germ cells and prepare the DNA for gel electrophoresis. Using detergents while stirring, you will attempt to break down the cell membrane, cell wall and nuclear membrane. Following this, the enzymes which might shred the long, thin fibers of DNA must be inactivated. If the procedure is done carefully, the DNA fibers will be released and can be seen and collected. Wheat Germ DNA Isolation Procedure: DAY 1 Gather all materials needed for the extraction Prepare solutions Weigh ingredients Become familiar with the lab protocol DAY 2 a. Place a plastic cup containing 100 ml distilled water into the water bath and, using a plastic spoon to stir gently, mix 1.5g wheat germ into the cup until it has dissolved as much as possible. Let the solution come to 55˙C, about 3 minutes. Add 5 ml full strength Palmolive detergent to disrupt the lipids in the cell wall prior to lysis. Maintain the temperature at 55˙C and stir occasionally for 5 minutes. Caution: Do not allow the temperature to exceed 60˙ C because this higher temperature could denature the normally double stranded DNA into single strands. Remove the cup from the hot water bath. Stirring constantly, dissolve 3g of meat tenderizer into the wheat germ solution (some will remain undissolved) and immediately add 10 ml of the baking soda solution. The tenderizer contains papain, a proteolytic enzyme (or protease) that degrades proteins such as nucleases. This, together with the 55˙C incubation, will degrade any nucleases or other proteins complexed to the DNA. Place the solution on ice for 15 minutes or store overnight in the refrigerator. If stored in the refrigerator, allow the solution to return to room temperature before continuing the procedure. b. c. d. DAY 3 a. b. c. Carefully pour approximately 20 ml of the lysate into a 50 ml Corning tube. Pour slowly so that any undissolved material remains behind in the cup. Using a serological pipet or a 10 ml graduated cylinder, carefully layer 10 ml ice-cold 95% ethanol or isopropanol over the lysate, forming a second layer. Use a glass rod (or Pasteur pipet with the tip sealed and bent to form a small hook) to spool the DNA which precipitates at the interface by passing the rod slowly through the two layers in a circular motion. Allow the excess ethanol to drip off onto a tissue. After most of the ethanol has dripped off, transfer the DNA to a 1.5 ml microcentrifuge tube or similar small tube, using the lip of the tube to scrape the DNA off the rod. If some residual alcohol is still present, use a micropipettor or small pipet to remove as much alcohol as possible or pulse spin in microcentrifuge and then remove the alcohol. Leave the tube open for 10-30 minutes or store overnight in a freezer without a cover to allow alcohol to evaporate. d. e. DAY 4 a. b. Depending on the amount of DNA you obtain, resuspend in 50 to 500µl sterile TE or another sterile buffer such as sterile saline. Vortex several minutes (or flick the tube vigorously or pipette) to solubilize the DNA. Vortexing is better because it is vigorous and solubilizing the DNA may take quite a while. (link to elecgtrophoresis/migration distance lab for gel instruction, and omit steps c-g) c. d. e. Each person will prepare a sample of DNA for loading into the gel by combining 3µl DNA, 15µl TE and 2µl loading dye into a 1.5 ml tube. Keeping track of lanes, load the entire 20µl into the well. When all samples have been loaded, close the lid on the gel box and attach the electrical leads, taking care not to jostle the box. Make sure that the power switch is off and the white dot on the rheostat is at the „min‟ position. Turn the power on and adjust the voltage to approximately 110V using the rheostat. Do not go above 110V or the gel will melt. Continue the electrophoresis for about 30 minutes or until the dye has reached midway between the second and third stripes. Then, turn down the rheostat, turn off the power supply and disconnect the leads. Wearing gloves, pour approximately 100 ml Methylene Blue Plus solution into a yellow staining tray. Transfer the gel from the gel bed to the tray by lifting the bed out of the gel tray and slowly sliding the gel from the bed into the stain. Make sure to label the tray with your group name. It is recommended that the gel be allowed to sit in the stain for 30 minutes, therefore the destaining process will be done for you so that the DNA bands will be visible for you tomorrow. (to destain - put 100 ml deionized/distilled water into a destaining tray. Using the spatula, remove the gel from the stain and transfer it to the destaining tray containing the water. f. g. DAY 5 (link to electrtophoresis/migration distance lab for photography instruction-omit steps if linked) a. b. c. d. Use the spatula to lift the gel from the tray, tipping the spatula slightly so excess water drains back into the tray. Transfer the gel to the surface of the light box, aligning the wells with the zero point on the ruler. Fit the hood of the camera over the gel, turn on the illuminator light and squeeze the shutter of the camera. Turn off the illuminator and pull the film from the camera by first pulling the protruding white tab to move the film out of the cassette. Next, grasp the black tab at the end of the film and slowly pull it out of the cassette. Wait 30 seconds and peel the picture (gray rectangle) from the backing. Each person should take a picture of the gel to keep for reference. Record the camera setting in your notes. Although the settings may differ from gel to gel depending on the extent to which a gel is destained, an “f” stop of 32 and a shutter speed of 15 (which is 1/15 sec) are reasonable settings to try first. Make sure you know how to change these settings to change the exposure. e. f. Laboratory Exercise: ISOLATION OF DNA FROM WHEAT GERM Teacher’s Prep DESIGNED FOR: Groups of 3-4 students TIME REQUIRED: 5/6 40 minute class periods PRELAB: Gel electrophoresis (optional /adds one day or combine with day 1 or 3) Each student will use a mock gel, a section of a gel containing several wells, to practice the loading process before loading the actual DNA samples into the first gel. MATERIAL LIST: Electrophoresis gel box apparatus Agarose Distilled water Hot plate/magnetic stirring bar Balance/scale Masking tape PREP HINTS: Weigh 0.4g agarose and place in a 150ml flask. Add 50ml distilled water or buffer to make a 0.8% gel. Double or triple this recipe depending on how many mini-gels you will be pouring. Pour agarose toward the side where you are going to put the comb. Pour only a small amount, and concentrate the agarose to the side. Let harden very slightly, and put on another layer, then add the comb. The gel should be very thin and not the full size of the gel block. If this is done ahead of time, place gels in a Ziploc bag in the refrigerator. Place the mini practice gel into a petri dish and cover with water. The gels shouldn‟t sit in the water too long. Let each group have their own gel, and make sure each student loads two wells. Student instructions: 1. Prepare a solution to use in practice loading by pipetting 180μl water into a small microcentrifuge tube and then adding 20μl of loading dye. Touch the pipet tip containing the dye to a meniscus of the water before expelling the dye. 2. Flick the tube vigorously with your finger to mix. Loading dye makes samples visible during loading and electrophoresis and contains a dense reagent such as glycerol to increase the density of samples so they will sink to the bottoms of the wells when loaded. 3. Spin the tube containing this solution for a few seconds in the microcentrifuge to bring the solution to the bottom. 4. Use your P20 micropipettor to transfer 20µl of the loading solution into four wells of the mock gel. To do this, first pick up 20µl of the solution with the micropipettor. Then, place the pipet tip just inside the top of the well or rest the tip against the upper edge of the well. Do not actually put the tip far into the well because the bottom of the well may puncture and cause the sample to leak out. 5. Dispense the dye solution slowly, allowing it to sink to the bottom of the well. Depressing the button of the pipettor too quickly could cause the sample to squirt out of the well. Try loading one “sample”. Do not load the rest unless you feel confident that you can load samples successfully. Ask me if you feel unsure of this process. MATERIAL LIST: Reagents & Solutions Recipes below: Sodium bicarbonate Detergent Protease Alcohol Buffer Methylene blue stain Deionized/distilled water Supplies & Equipment 7 oz. clear plastic cups 1.5 g raw wheat germ Thermometer Water bath at 55˙C Ice bucket and ice Balance/scale Vortex Weighing paper/boats/waxed paper Plastic teaspoons for mixing and weighing 3-10 ml serological pipets/10ml graduated cylinder Glass rods/Pasteur pipets Bunsen burner 50ml Corning tube 1.5 ml microcentrifuge tube staining tray/destaining tray vinyl gloves Polaroid camera/light box Spatula P20 micropipette/yellow tips Preparation of Solutions: Sodium bicarbonate (1N NaHCO3) buffer, pH 8.0: add 2g baking soda to deionized, distilled or tap water to bring volume to 25 ml (share between groups) Detergent: 5 ml full strength Palmolive liquid (or Wisk free) Protease: 3 g Adolph‟s 100% Natural Tenderizer (unseasoned) Alcohol for DNA precipitation: 10 ml 95% ice cold ethanol or isopropanol DNA Buffer: TE buffer (10 ml Tris, pH 8.0, 10mM EDTA) or sterile saline ISOLATION OF DNA FROM WHEATGERM: DAY 1:setting up stations/lab preparation MATERIAL LIST: Reagents Recipes below: Sodium bicarbonate Detergent Protease Alcohol Buffer Preparation of Solutions: Sodium bicarbonate (1N NaHCO3) buffer, pH 8.0: add 2g baking soda to deionized, distilled or tap water to bring volume to 25 ml (share between groups) Detergent: 5 ml full strength Palmolive liquid (or Wisk free) Protease: 3 g Adolph‟s 100% Natural Tenderizer (unseasoned) Alcohol for DNA precipitation: 10 ml 95% ice cold ethanol or isopropanol DNA Buffer: TE buffer (10 ml Tris, pH 8.0, 10mM EDTA) or sterile saline PREP HINTS: You can prepare these solutions a day or two ahead of the lab, or have the students prepare the solutions. Have each group of students responsible for making up one of the reagents for all of the groups. Each group should identify tubes with labels and/or permanent ink. Place sodium bicarb into 50ml Corning tube Pour 7ml Palmolive in 15ml culture tube Put 3g meat tenderizer in baggie/culture tube Pour 10ml purchased TE buffer/saline in 15ml culture tube Pour 10ml alcohol in 15ml culture tube Set up areas for the preparation of each of the solutions. Have tubes/pens/labels/scales and needed reagents/solutions at each area. If students are preparing the reagents, use the first day of the lab to familiarize them with the procedures and equipment. This has proven to be helpful and necessary. On day one, have student groups set up their stations with all of the needed equipment/solution. In the plastic cup, place the reagent tubes, and gather the pipets, glass rods/Pasteur pipets, Corning tube, and microcentrifuge tube. You can review each step of the lab and refer the students to the equipment in front of them. DAY 2: lyse cells (this is a good step to combine with day 3 if you have a lab period) PREP HINTS: If you do not have a water bath, you can improvise in a few ways; Place a large, shallow pot of water on a hot plate and monitor temperature Purchase a 10 gallon fish tank and a tank heater Run tap water that you have determined to be 55˙C into a Rubbermaid dishpan in the sink Make sure students hold onto plastic cup while in the bath – they float! Use small thermometer to stir wheat germ. This is much easier than monitoring temperature and stirring with a spoon. Ice can be placed in a Rubbermaid dishpan or a Styrofoam container. Bait buckets work well, and are inexpensive and found at any of the “big box” stores. Check for ice from the athletic trainer, nurse or cafeteria. It‟s a good idea to ice the solution even if you are placing it in the refrigerator overnight. Place the plastic cup inside Glad sandwich bag, and place in the refrigerator overnight. DAY 3: extract DNA This is a good day to practice loading DNA into the mini-gels since it doesn‟t take very long to spool DNA. The electrophoresis lesson fits well here as the DNA will be loaded into the gel on day 4. PREP HINTS: Have the tubes of alcohol chilling on ice before the students arrive in class You can speed the drying of the DNA in the tube by gently blowing air from a hair dryer into the tube. Do this if you want to continue to the day 4 exercise on the same day. DAY 4: solubilize DNA and load gel **if you are going to cut the DNA, refer to the restriction enzyme lab part 1-C. PREP HINTS: Generally, there is quite a bit of DNA. You will be adding quite a bit of saline in order to resuspend the DNA. If the DNA is still pelletized after a minute or two of vortexing, you need to add more saline. Make sure that students keep track of the lanes they load. There should be enough DNA for the students to prepare and load two samples each. Keep the staining tray/identification marker next to the gel box so gels do not get mixed up between groups. Do not keep gels near a window while destaining. The sunlight will completely fade the dye so that the DNA is invisible. After the initial 30 minute destaining period, cover gels with distilled or tap water and leave overnight. LAB TIPS: Purchase raw wheat germ that is relatively fresh. Hodgson‟s Mill wheat germ has given great yield. Gels can be poured at any time before day 4. Place gels in plastic Ziploc bags in the refrigerator with a little buffer to keep them moist. Keep the combs in the wells until you fill the gel box with buffer right before you‟re ready to load the genomic DNA. Demonstrate how to use the vortex. Students tend to press down too hard and rub the “nubs” off of the platform. GRADING IDEAS: Prepare a narrative lab, including the Polaroid photograph of the gel. Assess lab techniques each day while students are working in class. DISCUSSION QUESTIONS IF USING RESTRICTION ENZYMES 1. 2. What does the 65C incubation step at the end of the restriction digest accomplish? What would be the consequence of omitting this step? Compare the undigested and digested genomic DNA samples. Draw a representation of the gel, and discuss the results in each of the lanes. Explain if this is an expected or unexpected result. Using the kilobase ladder standard as a guide, what is the approximate size of the DNA molecules in your digested and undigested genomic DNA samples? Knowing that chromosomal DNA molecules are extremely large (millions of base pairs in size), explain the smear of DNA in the undigested sample. 3.