DNA Extraction from Strawberry
Why extract DNA from a strawberry? Demonstrate that food actually has DNA in it! One study showed that some people think that traditionally bred plants DON T have DNA while biotech-developed plants do Show that DNA is a physical entity that can be analyzed, cut into pieces and reassembled People have a hard time visualizing DNA. This makes it very difficult for them to understand how transformation works, etc. Demonstrate that DNA from various sources looks the same Important to understand that, even though DNA comes from different species, it has the same basic makeup (bases, base-pairs, etc.) Use as an attention-getting activity and to increase participant involvement
DNA Lab objectives Life Science The cell - DNA is located in the cell nuclei of all living organisms Molecular basis of heredity - DNA molecule directs the synthesis of every protein and contains all the genetic information that is passed on to new cells. Physical Science Chemical reactions - structure of cell membrane Science as Inquiry Design and conduct scientific investigations Formulate and revise scientific explanations and models using logic and evidence
onion strawberry banana peas broccoli Calf thymus University of California Davis Partnership for Plant Genomics Education 2007 kiwi Wheat germ
Onion extraction found at: http://www.accessexcellence.org/ae/atg/data/released/0508- LanaHayes/onion.html. Use center of onions. Chop in blender. Add meat tenderizer before or after extraction buffer One of the reasons strawberries work so well is that they are soft and easy to pulverize. Also, ripe strawberries produce enzymes (pectinases and cellulases) which aid in breaking down the cell walls. Most interestingly, strawberries have enormous genomes. They are octoploid, which means they have eight of each type of chromosome (which equals abundant DNA).
Three Main Steps in Any Extraction Break apart cells Release DNA from nucleus precipitation of the DNA
Strawberry DNA Extraction Activity To prepare material for 30 teams of two Obtain the following items: 30 - One quart Ziplock bags 30 - #2 coffee filters 30-5 oz plastic cups 15 - Strawberries 1 - Teaspoon table salt (2 grams) 1 - Bottle of Suave soap (with EDTA) 1 - Pint bottle of 91% isopropyl alcohol 1 500 ml bottle to mix ingredients in 1-50 ml graduated cylinder 30-15 ml test tubes 30 50 ml test tubes 30-1 ml disposable pipettes 1 teaspoon for measuring salt
Protocol 1. Place one strawberry in a zip lock baggie. 2. Smash strawberry with fist for 1-2 minutes. 3. Add 5 ml extraction buffer to the bag. 4. Mush again for one minute. 5. Filter through cheesecloth in a funnel into tube (1/4 full) 6. Slowly pour the ice-cold alcohol into the tube until the tube is half full. 7. At the interface, you will see the DNA precipitate out of solution and float to the top. 8. Spool DNA on your glass rod or pipette tip.
DNA extraction buffer DNA extraction buffer 100ml of shampoo (without conditioner) 15g NaCl 900ml water Alternatively 50ml dishwashing detergent 15g NaCl 950ml water
Why Detergent? Detergent dissolves the lipids in the cell membranes and nuclear envelope Soap molecules and the lipids (fats) in cell membranes are made of two parts: hydrophillic heads hydrophobic tails When detergent comes close to the cell, it captures the lipids and proteins (due to their similar structures)
Plant cell membrane- phospholipid bilayer University How of California detergent Davis disrupts Partnership the for Plant plant Genomics cell Education membrane 2007
Why Salt? Salt stabilizes the negatively charged phosphate groups and allows the DNA strands to clump together. DNA has a negative electrical charge due to the phosphate groups on the DNA backbone, and the electrical charge makes it soluble. When salt is added to the sample, the positively charged sodium ions of the salt are attracted to the negative charges of the DNA, neutralizing the electrical charge of the DNA. This allows the DNA molecules to come together instead of repelling each other. University of California Davis Partnership for Plant Genomics Education 2007
Why Ethanol or alcohol? The addition of the cold alcohol precipitates the DNA since it is insoluble in high salt and alcohol. Since water is soluble in ethanol, the water becomes dissolved into the comparatively large volume of ethanol, which effectively removes the water from around the DNA causing the DNA to precipitate.
Protocol 1. Place one strawberry in a zip lock baggie. 2. Smash strawberry with fist for 1-2 minutes. 3. Add 5 ml extraction buffer to the bag. 4. Mush again for one minute. 5. Filter through cheesecloth in a funnel into tube (1/4 full) 6. Slowly pour the ice-cold alcohol into the tube until the tube is half full. 7. At the interface, you will see the DNA precipitate out of solution and float to the top. 8. Spool DNA on your glass rod or pipette tip.
How Do I Begin? Place the strawberry in a zip lock bag and remove the air Squash the berry with your hand to begin the process
Soap & Detergent By adding a small amount of table salt (NaCl) to the soap solution, the solution can punch holes in the nuclear and cell membranes The soapy solution also helps removes proteins
Extracting the DNA The contents of the cell (organelles, proteins, etc.) must be separated from the DNA The larger cell parts can be removed by filtering the solid from the liquid
Collecting the DNA The DNA will appear as a white precipitate once the alcohol is added HOLD THE TUBE by the TOP, not the bottom so the DNA strands won t fragment from the heat of your hands!
Spooling the DNA DNA is sticky and will adhere to other surfaces A glass stirring rod A glass stirring rod can be used to spool (remove) the DNA by using a turning motion
Discussion Questions Where can DNA be found in the cell? What is DNA? What are the three basic steps in any DNA extraction? What is the purpose of the detergent? Of the sodium chloride? Of the alcohol? Describe the appearance of your final product. Is this a pure DNA sample? List some practical applications where extracting DNA is necessary.