Strawberry DNA Extractions Objectives: Participants will be able to….. Explain how DNA is present in the cells of every living thing. Use an extraction procedure to isolate DNA from the cells of a strawberry Examine the visual characteristics of a large amount of DNA precipitate. Take home a sample of extracted strawberry DNA. Background: Deoxyribonucleic acid (DNA) is the chemical inside our cells that contains the genetic code. Each DNA molecule is a long polymer of smaller chemical units called nucleotides. There are 4 different types of nucleotides nicknamed A,T, G and C. These occur in a particular sequences along the length of the DNA molecule in each individual’s cells. The order of the nucleotide sequence is slightly different in each organism in a particular species, making that individual unique. The order of nucleotides in DNA is what forms the genetic code. The instructions in the genetic code tell the embryo how to divide and develop into each particular organism and how each cell should carry out the biochemical reactions that keep us alive and allow our bodies to function. All living things are made of cells and each cell contains a full set of genetic instructions in the form of several long molecules of coiled DNA called chromosomes. If the DNA molecules from each chromosome were uncoiled and laid end to end, each cell in a human would have a total of nearly 6 feet of DNA. This DNA is very neatly supercoiled into chromosomes and packaged into a compartment called the nucleus in every cell. The procedure we will use today will allow the DNA to escape the boundaries of the nucleus and the cell membrane and precipitate into a visible form we can observe with the naked eye. The white fibers we will see in our preparation today are actually thousands of DNA strands wrapped around each other. An individual DNA strand is so small, it can only be imaged by the most sophisticated and specialized electron microscope. Scientists who study DNA and the gene sequences contained in the DNA molecules begin their study with an isolation procedure similar to the one you are performing today. They would follow up by additional purification steps to remove all the protein and contaminating materials (including enzymes that can attack and destroy DNA in storage unless removed). They would then use additional procedures to isolate the particular region of DNA that contains the genes (DNA segments) they are intending to study. Procedure notes: Today we will isolate DNA from strawberry cell. Ripe fruit is a good material for isolating plant DNA since the cell walls are already weakened by the ripening process. Commercial strawberries 8 sets of each type of chromosome (this is called octoploid). Wild strawberries have only 2 sets of chromosomes (diploid). This makes grocery store strawberries a particularly good material to use. The procedure used today must accomplish several things. First, the cells must be broke open. The nuclear membrane must also be broken open and the DNA allowed to escape into the extraction buffer. The cell and nuclear membranes are made of fats and proteins. These are chemically very different from the nucleotide chemical that makes up DNA. The detergent and salt in the extraction buffer do a good job of separating the proteins and fats from the nucleus and the rest of the cell contents. The DNA is dissolved in the water in the extraction buffer and needs to be precipitated so we can see it. This is accomplished by using alcohol. By carefully placing the alcohol layer on top of the detergent layer, the proteins and fats in the cell debris will stay in the lower layer and the DNA will rise to the top and form insoluble threads in the alcohol layer. By using a tool such as a bamboo skewer to carefully wind up the DNA threads, you can lift the DNA out of the test tube, and remove it for study. The DNA can be dried and re-dissolved in water, or kept as a precipitate in alcohol in a closed tube. Illustrations of experimental apparatus Extraction set up Precipitation set up Materials Ripe strawberry Ziploc bag (quart size) Small beaker or glass tumbler Small funnel coffee filter trimmed to fit funnel (As an alternative to a funnel: Use an accordion style coffee filter and use rubber band to secure to top of glass container) 15 ml. clear glass or plastic centrifuge tube with cap graduated cylinder or liquid measuring cup non-iodized salt (iodized salt will work fine) distilled water 95% ice cold ethanol (can use Everclear liquor, but may need to modify amounts to see a DNA precipitate) Ivory, Dawn or Palmolive liquid dishwashing detergent (minimal additives) 6 “ square of waxed paper 1.5 mL Eppendorph test tube with snap lid (optional) Extraction buffer solution recipe 50 mL liquid detergent 15 g salt add 950 mL distilled water for a total of 1000 ml Use 10 mL of extraction buffer per large strawberry Protocol 1. 2. 3. 4. Select one medium to large ripe strawberry (approx. 30 gm.) and remove stem. Place inside Ziploc bag. Press out as much air out of the bag as possible and seal it. Gently mash the strawberry in the bag for 2 minutes until large chunks are no longer visible. 5. Add 10 mL extraction buffer to the bag, press the air out and reseal the bag. 6. Mash the strawberry and buffer mixture for 1 minute taking care to avoid bubbling and foaming as much as possible. 7. Keep foaming to a minimum if possible. 8. Place funnel with coffee filter into beaker. 9. Pour liquid contents of Ziploc bag into filter. 10. Collect filtrate in beaker 11. Pour approximately 3 mL filtrate into test tube (2 cm. or 1/8th tube volume if tube has no volume markings.) Try to avoid pouring foam into tube. 12. Obtain 9 mL ice cold 95% ethanol. 13. Pour ethanol slowly down side of tube, taking care not to disturb interface between extraction bufferlayer and alcohol layer . 14. Note the white thread-like material that accumulates at the interface between the alcohol and buffer layers. This is DNA. 15. Let the mixture sit undisturbed for a minute or two. 16. The DNA will float in the alcohol. The DNA of the strawberry cells will be long threads that easily spool. 17. Use a wooden bamboo skewer or plastic inoculating loop to carefully spool the DNA from the interface and remove it from the tube onto the waxed paper square. 18. Observe the visible characteristics of the DNA once removed from the solution. 19. The DNA can be allowed to dry to a near transparent film on the waxed paper, or…. 20. If desired, the DNA mass can be placed in a 1.5 mL plastic tube while still wet and covered with alcohol for safekeeping.