Gel Electrophoresis Experiment Fall 2008

1. Gel Electrophoresis Experiment Fall 2008 A Timed Presentation - do not click the mouse. Approximate Run Time - 4 minutes

2. Gel Apparatus Comb produces 28 wells Gel is 22.5 x 22.5 cm in size

3. Restriction Enzyme Digest Oven Temperature accuracy is good to + or - 1 degree celsius

4. Protocol Uncut: DNA 18.75 μl 10 x Buffer 15 μl Water 116.25 μl 6 x Loading Buffer 30 μl 30 μl/well – approximately 1.25 μg DNA/well Each sample of DNA was incubated for 1 hour at 37°C. Each sample and the ladder was then loaded into each well as outlined in the following slides. Single cut: DNA 37.5 μl 10 x Buffer 22.5 μl BamHI 15 μl Water 150 μl 6 x Loading Buffer 45 μl 30 μl/well – approximately 1.67 μg DNA/well BamHI 900 bp pBabe/cpp32 6000 bp EcoRI Bouble cut: DNA 37.5 μl 10 x Buffer 22.5 μl BamHI 11.25 μl EcoRI 11.25 μ l Water 142.5 μl 6 x Loading Buffer 45 μl 30 μl/well – approximately 1.67 μg DNA/well 5100 bp pBabe plasmid donated by Dr. Tang

5. Lane 28 Lane 20 28 lane gel Lane 10 Lane 1

6. Carmen loading lane #2; 1st of the uncut plasmids

7. Emily loading lane #3 2nd lane of the uncut plasmid

8. Kristin loading lane #4 3rd lane of the uncut plasmid

9. Alma loading lane #5 4th lane of the uncut plasmid

10. Doug loading lane #6 5th lane of the uncut plasmid

11. Danny loading lane #7 6th and last lane of the uncut plasmid

12. Alicia loading lane #8 1st of the single RE cuts

13. Bryanah loading lane #9 2nd of the single RE cuts

14. Megan loading lane #10 3rd of the single RE cuts

15. Brittany loading lane #11 4th of the single RE cuts

16. Alma loading lane #12 5th of the single RE cuts

17. Carmen loading lane #13 6th of the single RE cuts

18. Emily loading lane 14 7th of the single RE cuts

19. Aaron loading lane 15 8th of the single RE cuts

20. Doug acquiring DNA sample

21. Doug loading lane 16 9th of the single RE cuts

22. Krsiten loading lane 18 1st of the double RE cuts

23. Danny loading lane 19 2nd of the double RE cuts

24. Alicia loading lane 20 3rd of the double RE cuts

25. Megan loading lane 21 4th of the double RE cuts

26. Brittany loading lane 22 5th of the double RE cuts

27. Alma loading lane 23 6th of the double RE cuts

28. Emily loading lane 24 7th of the double RE cuts

29. Aaron loading lane 25 8th of the double RE cuts

30. Alma loading lane 26 9th of the double RE cuts

31. Time: 0 The gel has been loaded and is ready to have the power turned on

32. Time: 40 minutes Note the movement of the DNA (light blue - due to stain) and the buffer stain (dark blue)

33. Time: 2 hours 20 minutes Note the large distances between the DNA (light blue) and the buffer stain (dark blue)

34. Time: 3 hours 40 minutes Note the differences in the distances the different DNA samples have moved 3 Buffer Stain 2 1

35. DNA Ladder used in the gel electrophoresis experiments

36. 27 17 Ladder Rows: 1

37. Uncut Plasmid Rows 2-7

38. Single cut plasmid Rows 8-16

39. Double cut plasmid Rows 18-26

40. 9000 bp 8000 bp 7000 bp Ladder Marks

41. 6000 bp 5000 bp 4000 bp Ladder Marks

42. 3000 bp 2000 bp 1650 bp Ladder Marks

43. 1000 bp 850 bp 650 bp Ladder Marks

44. 3000 BP 4000 BP 5000 BP 1650 BP 2000 BP 6000 BP 7000 BP 8000 BP Note DNA ladder from this view

45. Uncut plasmid Runs more as a smear between 3250 and 4000 bp in length

46. Single cut plasmid Falls at approximately 6000 bp in length

47. Double cut plasmid shows two distinct bands at 5000 bp and 900 BP in length

48. The SBI4U class of Hill Park Secondary School wish to thank Dr. D. Tang (McMaster University/St. Joseph’s Hospital) and his lab technician for donating the DNA, Restriction Enzymes and Ladder and for calculating the amounts of material required to complete this procedure successfully.