Genes in a Bottle DNA Extraction

Relevance of DNA isolation Isolation of DNA is often the first step before further analysis DNA profiling cloning disease diagnosis DNA sequencing genetically modified organisms (GMO) - agriculture, pharmaceutical Environmental testing, biodefense

What are the Structures of the Cell?

Cell Structures Nucleus- Contains nuclear DNA (genome) *Strawberries are octaploid (8n)

Cell Structures Chloroplasts & Mitochondria contain their own DNA

Cell Structures Lysozomes contain digestive enzymes

Protocol Highlights: Genomic DNA Extraction Collect cells Add extraction buffer (soap) to cells to break open cell and nuclear membranes and release nuclear contents Digest sample with protease (meat tenderizer) to degrade proteins. This step isn’t necessary for all cell types Precipitate DNA with cold alcohol in high salt

Laboratory Protocol 1.Obtain your sample & place in a zipper sealed bag. 2.Smash for 2 minutes. If the bag breaks double bag it. 3.Add extraction buffer to the bag & mush for another minute. 4.Pour the extract into the filtering apparatus & let it drip directly into the test tube. Only fill the test tube about 1/8 full. You can fill one for each person in your group.

Laboratory Protocol 5. SLOWLY POUR COLD ETHANOL (FROM SQUIRT BOTTLE) INTO THE TUBE SO THAT THE TEST TUBE IS HALF FULL WITH LIQUID 6. Let stand for A FEW MOMENTS. 7. DIP THE LOOP OR ROD INTO THE TUBE RIGHT WHERE THE ALCOHOL AND EXTRACT LAYERS ARE IN CONTACT WITH EACH OTHER. KEEP THE TUBE AT EYE LEVEL SO YOU CAN SEE WHAT IS HAPPENING.

Genes in a Bottle Why Perform Each Step?

Squishing the sample begins to break the cells apart. Since the fruit/vegetable is ripe, lysosomes within the cell are already releasing digestive enzymes (pectinases and cellulases), which are already breaking down the cell walls & cell, nuclear & organelle membranes. Cell Collection

Step 2: Extraction (Lysis) Buffer? soap & NaCl solution *T he detergent in the shampoo helps to dissolve the phosholipid bilayers of the cell membrane and organelles. * Na + ions of NaCI bind to the phosphate groups of DNA molecules, neutralizing the electric charge of the DNA molecules. * The addition of NaCI allows the DNA molecules to come together instead of repelling each other, thus making it easier for DNA to precipitate out of solution when alcohol is added.

Na + O CH 2 O P O O O Base CH 2 O P O O O Base OH Sugar O DNA structure

Adding Ice Cold Alcohol? DNA does not dissolve in alcohol. The addition of cold alcohol makes the DNA clump together and precipitate out of solution. Precipitated DNA molecules appear as long pieces of fluffy, stringy, web-like strands. Microscopic oxygen bubbles “aggregate”, or “fuse” together, as the DNA precipitates. The larger, visible air bubbles “lift” the DNA out of solution, from the aqueous into the organic phase.

Prepare stoppers for necklaces 1.Remove stopper from vial 2.Trim “ears” off stopper on 2 sides 3.Replace in vial for later necklace creation WHY? So the stopper doesn’t create an air pocket preventing the cap from seating fully on the vial

Preserving DNA Sample: DNA Necklace Preparation 1.Using a disposable plastic transfer pipet, carefully transfer the fluffy DNA strands you extracted into the small glass vial. –Transfer as much of your DNA and as little alcohol as possible. –The vial should be filled no higher than 2 mm from the top of the neck of the vial.

Preserving DNA Sample: DNA Necklace Preparation 2.Firmly push the trimmed plastic stopper cap into the neck of the vial to seal the glass vial. 3.Slip the waxed cord through the silver cap. 4.Apply a small drop of super glue to the inside of the silver cap.

Preserving DNA Sample: DNA Necklace Preparation 5.Place the silver cap onto the top of the glass vial and press down firmly for 30 seconds. Allow the glue to dry for an additional 10–15 seconds, and then check for a complete seal. 6.After the glue has dried, tie the waxed cord.

Genes in a Bottle Kit Congratulations! You have just created your very own DNA Necklace!

How Long Does the DNA in the Necklace Last? The DNA in the glass vial can last for years. Add more alcohol into the vial if some evaporation occurs.