DNA Isolation, Restriction Enzyme, Digestion, and Gel Electrophoresis

1. Write your group # on the test tube 2. With graduated cylinder, get 10-15ml of detergent/buffer solution from the refrigerator and add it to your test tube 3. Stir vigorously with the large glass stirring rod. Be careful not to break the rod or test tube 4. Answer the first question on page Place the test tube in the warm water bath for 20 minutes.

 What does DNA stand for? Deoxyribonucleic acid  Where do you find DNA? In the nucleus  What is DNA? Genetic information  What do we use DNA testing for? Paternity, crimes, identification, etc  Where can you get DNA samples? Blood, hair, saliva, skin, etc

 What is DNA made of? Nucleic acid (the 4 th carbon compound) Two strands of nucleotides  Sugars, phosphates, nitrogenous bases  What are the nitrogenous bases? Adenine Cytosine Guanine Thymine  Which nitrogenous bases associate? Adenine – Thymine Cystosine – Guanine

 Restriction enzymes – enzymes that break down DNA at certain sequence  Eco RI cuts at GAATTC

1. Gently stir test tube with large glass rod 2. Place a funnel into the 50ml beaker 3. Place a piece of cheesecloth over funnel 4. Pour your test tube solution into the funnel and allow to filter into beaker (need 10-15ml of solution) 5. Remove funnel and cheesecloth 6. With graduated cylinder, get 5ml of meat tenderizing solution from the refrigerator and add to the beaker 7. Use the small stirring rod to mix GENTLY 8. Allow mixture to sit on your desk for 5 minutes 9. Answer questions in the book while you wait

10. With graduated cylinder, get 10-15ml of 95% ethanol. 11. Slightly tilt your beaker while SLOWLY adding ethanol 12. Wait 2 minutes 13. Place the small glass rod straight down into the beaker until it touches the bottom 14. Gently twist the rod in one direction. A white thread-like precipitate (DNA) will wrap around the glass rod as you stir 15. Continue turning until you have spooled the DNA onto rod 16. Carefully tilt the rod and lift out the DNA 17. Hold the glass rod in the air for 1 minute to let the DNA dry 18. While waiting, another team member needs to get a tiny test tube and add about 1/3 full or 1” of water. 19. Add DNA to small test tube

20. Send ONE team member up front to learn how to pipette. (Clean up while your classmates are learning how to pipette. DO NOT throw away your DNA) 21. You have a small blue tube and a small yellow tube. Label the top of the lid. Blue is “A” and yellow is “B” 22. On the side of each tube write your group # “G1” and your section. Mon “D”, Tues “H”, Wed “L”, Thurs “Q” 23. Pipette 18µl of DNA solution into blue tube and 18µl into yellow tube. 24. Use pipette tip to mix 25. Bring tube A (blue) to me for restriction enzyme buffer and enzyme 26. Place tubes into the incubator

 Exercise 8 - Record appearance and root/stem measurements and replace solutions if needed. Page 64  Exercise 7 – Record height of each plant on Page 58 for both wild type and rosette. Add one drop of water to each leaf of your “A” plants and add one drop of gibberellic acid to each leaf of your “B” plants