Gel Electrophoresis Experiment Spring 2005

1. Gel Electrophoresis Experiment Spring 2005 A Timed Presentation - do not click the mouse. Approximate Run Time - 2 minutes 38 seconds

2. Gel Apparatus

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

4. Restriction Enzyme Digest Oven

5. Mixing the Loading Buffer

6. Loading DNA

7. Loading DNA

8. Loading DNA

9. Loading DNA

10. Loading DNA

11. Loading DNA

12. Loading DNA

13. 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

14. 20 minutes 80 Volts

15. 30 minutes 80 Volts View from above Note die markers

16. Note separation of die markers 30 minutes 80 Volts

17. 55 minutes 80 Volts View from above Note separation of die markers

18. 55 minutes 80 Volts Note separation of die markers

19. DNA Ladder used in the gel electrophoresis experiments

20. Ladder Rows: 1 8 18 28 Normal light 55 minutes; 80 Volts 17 hours; 30 Volts 1 hour ; 80 Volts

21. Ladder Rows: 1 8 18 28 Background light

22. 2 To 7 Uncut Plasmid Rows Normal light

23. 2 To 7 Uncut Plasmid Rows Background light

24. Single Digest Rows 9 to 17 HindIII Normal light

25. Single Digest Rows 9 to 17 HindIII Background light

26. Double Digest Rows 19 to 27 Normal light

27. Double Digest Rows 19 to 27 Background light

28. 400 bp

29. 850 bp

30. 1650 bp

31. 2000 bp

32. 5000 bp

33. 6000 bp

34. 12000 bp

35. No cut (enlarged) 2000 bp 3000 bp 4000 bp 5000 bp 8000 bp 12000 bp Uncut, circular plasmid runs as a smear slightly smaller than the size of the actual number of base pairs. The smear is seen mainly in the 3000 – 4000 bp length.

36. Single cut (enlarged) 2000 bp 5000 bp 6000 bp Single cut (BamHI) plasmid runs at the 6000 bp length.

37. Double cut (enlarged) 850 bp 2000 bp 5000 bp 12000 bp Double cut (BamHI; EcoRI) plasmid runs at just over the 5000 bp length and a second smaller piece at approximately 850 bp.

38. 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.