DNA Extraction from Tomatoes DNA is the genetic information inside of cells. Every living organism is comprised of cells has DNA that provides the instructions for making proteins needed for survival. When specific sections of DNA called genes are turned on, gene expression is said to occur. As different genes are expressed, different characteristics appear in the cells and the organism as a whole. The process of turning genes on during gene expression first involves http://www.graphic- production of RNA molecules and then production of design.com/Web/Critique/tomato.html proteins that act as enzymes, messenger molecules (hormones), and structural components within the cell and organism as a whole. Think about tomatoes. A fresh ripe juicy summer tomato stimulates your taste buds in all the right places. But in the winter tomatoes tend to be tougher and tasteless. Both types of tomatoes are fruits that come from the same species of tomato plant. Because they are members of the same species, they have the same amount of DNA and the same types of genes although it is possible the genes have different alleles. The summer tomato is ripe and sweet, while winter tomato is not fully ripe and has not had the opportunity to develop the same flavor characteristics as the summer tomato. These differences are due to gene expression. While the DNA in both tomatoes is the same, the summer (fully ripe) tomato has genes being expressed that lead to sugar production for sweetness, red pigments for coloration, and enzymes for destruction of green chlorophyll. The longer the fruit is on the plant, the more fully the genes are expressed. Gene expression within cellular DNA is important for differentiation (specialization of cells) in every multicellular organism. As well, single cellular organisms may use gene expression to turn genes on and off to match specific environmental conditions around them. Materials: Fresh tomatoes (1 wedge/ group about 3cm at widest point) Extraction Solution: Salt, Soap (shampoo), water (20ml per group) Ziploc Bags (1 per group) Cheesecloth or #2 coffee filter (1 per group) Styrofoam Cup (1 per group) 50 mL Tubes or Beakers (1 for each group) 10 mL Graduated Cylinder (1 per group) Cold Alcohol (5-10ml per group) http://www.eatcatomatoes.org/ 3x5 Card (1 per Group) Consumer/Content.aspx?id=62 Methylene Blue (2-3 Drops) www.dnamnd.med.usyd.edu.au/DNA_logo.jpg Procedure: 1. Place tomato piece in a Ziploc Bag 2. Add your 20mL of the Extraction Solution to the Ziploc bag. Carefully remove as much air as possible without spilling the liquid out of the bag and zip the bag closed. Squeeze the tomato pieces and solution inside the bag with your fingers or beat the tomato pieces with the bottom of a blunt object for about 5 minutes. Be sure not to puncture or rip the bag during the process. Questions - Place your answers on the student answer sheet. What does squeezing or beating do to the cells that make up the tomato? The Extraction Solution in the bag is made up of soap, salt, and water. What do these ingredients do to the cells that make up the tomato? Remember the pre-lab… Take a small amount of the tomato and Extraction solution mixture and prepare a wet-mount slide. Observe on low power then switch to high power and draw cells after they have been lysed. What is lysis? 3. Filter the mixture through the cheesecloth or coffee filter. Be careful to allow only the liquid and not chunks of tomato that remain into the Styrofoam cup. Question - Place your answers on the student answer sheet. Is the tomato cell DNA being trapped in the filter or moving through the filter in this step of the procedure? 4. Measure 10 mL of the filtered tomato solution using a graduated cylinder and place in tube or beaker. 5. Add 10 mL of cold alcohol to the 50 mL tube with the tomato solution in it and start timing. Do not shake or swirl the tube. Watch the tube closely and observe the DNA as it comes out of the solution and forms a condensed mass within the tube. Record the changes that occur inside of the tube and the amount of time it takes for each change to occur. Questions and Observations - Place your answers on the student answer sheet. Describe what happened to the DNA solution when the cold alcohol was added. In doing so, accurately describe the changes that occurred and give the amount of time it took for each change to occur. If the alcohol had not been cold, do you suppose the changes in the tube would have occurred more slowly or more quickly? State your hypothesis and give a reason for your idea. 6. Using a plastic spoon, carefully scoop the DNA out of the tube or beaker and place it on a paper 3x5 card. Add 2 or 3 drops of methylene blue to completely cover the DNA and let the DNA sit undisturbed overnight. Questions - Place your answers on the student answer sheet. Did the DNA on the card stain blue? _________ What parts of the tomato cells stained blue on the wet-mount prepared in the pre lab? How dies this correspond to the stained DNA? DNA Extraction - Student Answer Sheet Name ______________________ Questions from Step 2 What does squeezing or beating do to the cells that make up the tomato? The Extraction Solution in the bag is made up of soap, salt, and water. What do these ingredients do to the cells that make up the tomato? Drawing: What is lysis? Question from Step 3 Is the tomato cell DNA being trapped in the filter or moving through the filter in this step of the procedure? Questions and Observations from Step 5 Describe what happened to the DNA solution when the cold alcohol was added. In doing so, accurately describe the changes that occurred and give the amount of time it took for each change to occur. If the alcohol had not been cold, do you suppose the changes in the tube would have occurred more slowly or more quickly? State your hypothesis and give a reason for your idea. Record the changes that occur inside of the tube and the amount of time it takes for each change to occur. Questions from Step 6 Did the DNA on the card stain blue? _________ What parts of the tomato cells stained blue on the wet-mount prepared in the pre lab? How does this correspond to the stained DNA? Summary Questions 1. Make a list of descriptive words that could be used to explain what DNA looks like to someone who has never seen DNA before. 2. If you could repeat this experiment several times, what other substances would you choose to try and get DNA? Explain your reasoning and thinking. Teacher Notes Tubes Corning - Cat #430897 - 50mL Centrifuge Tube, Self-Standing – Case of 500) Corning® 50 mL Centrifuge Tubes Extraction Solution (500mL) Mix 50 mL of Shampoo and 7.5g Salt. Dissolve salt by stirring slowly to avoid bubbles/foaming. Add water to a final volume of 500 mL. Measure 20ml per group. Note1: Do not use shampoos with conditioner or baby shampoo. Note2: Suave and White Rain shampoo work well. The detergent dissolves the lipids that comprise the cell membranes. After cellular membranes are disrupted, the DNA and cell components are present in the solution. The detergent causes lipids and some proteins to precipitate out of solution, leaving DNA and some proteins. The salt will ionize in solution to form Na + and Cl- ions. These ions bind with DNase enzymes that will degrade the DNA and serve to help protect the DNA during the extraction process. As well, Na + will interact with the negatively charged regions on the DNA so that strands of DNA will come together during the extraction process. Alcohol - Cold 95% Ethanol or Isopropanol (rubbing alcohol) – Place in Freezer Prior to Use The alcohol acts to remove water from around the DNA. Alcohol forces the water away from the DNA and causes it to precipitate into a wad. The alcohol must be cold for optimal results, otherwise the alcohol and water will mix more readily and the water will not be drawn away from the DNA. As a result, the DNA will not precipitate. Methylene Blue Methylene Blue stains the Nucleus and Cytoplasmic Granules of cells blue and will stain the DNA extracted from tomatos blue. Hydrophilic Molecule – Basic (+ charge) will associate with acidic R-groups of protein molecules and the sugar phosphate backbone of DNA (- charge). Methylene blue will also intercalate with the base pairs in the inner region of the DNA since it is a 3-ringed flat molecule. As well, methylene blue will turn colorless when reduced and can be used as an indicator for REDOX reactions - peak absorbance at wavelengths of 609nm and 668nm when blue. Preparation - Mix 1g in 25ml of dH2O. Note: If mixed in ethyl alcohol, it will dehydrate DNA. Extensions – Other Staining Procedures Iodine – Stains the Nucleus, Cilia, and Flagella of Cells Iodine will not stain the DNA. It will remain brown/amber and not change to purple, blue, or black. This shows that there is no starch (polysaccharide) associated with the DNA. Acetocarmine – Stains the Nucleus / Chromosomes / DNA Acetocarmine will stain the DNA red and is a common stain for the nucleus cells. Acetocarmine is a basic dye with a positive charge that is attracted to the negative charge of the DNA. Note the precautions on the acetocarmine stain and use appropriate lab safety precautions. Bradford Reagent – Indicator molecules conformationally change when exposed to protein. The shift in structure changes the wavelengths of light absorbed and reflected by the molecule. As a result, it shifts from grayish-blue to bright blue. Bradford Reagent 100mg Coomassie Brilliant Blue G-250 Dissolve in 50ml of 95% Ethanol Add 100ml Phosphoric Acid (85% w/v) Dilute with Distilled Water to a final volume of 1 Liter. Note – Be sure to get Coomassie Brilliant Blue G –250, not R-250. Aldrich Chemical Co. PO Box 355 Milwaukee, WI 53201 (414) 273 – 3850 Bradford reagent stains proteins and turns from grayish blue to bright blue. All eukaryotic DNA will stain blue with this reagent since it is associated with histone proteins. This can be a valuable technique to see that eukaryotic DNA is actually comprised of both DNA and protein. Ask students if DNA from a prokaryotic cell (no histones) would also stain blue with Bradford reagent.