1. Introduction to Gel Electrophorsis

2. Model of DNA DNA is Comprised of Four Base Pairs

3. Deoxyribonucleic Acid (DNA)

4. DNA Schematic O O Phosphate Sugar Phosphate Sugar O O P O Base O CH2 OOH

5. DNA Restriction Enzymes
• Evolved by bacteria to protect against viral DNA infection
• Endonucleases = cleave within DNA strands
• Over 3,000 known enzymes

6. Enzyme Site Recognition
Restriction site
Palindrome
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences
(eg GAATTC)
Fragment 2
Fragment 1

7. 5 vs 3 Prime Overhang
Enzyme cuts
• Generates 5 prime overhang

8. Common Restriction Enzymes
EcoRI
– Eschericha coli
– 5 prime overhang
Pstl
– Providencia stuartii
– 3 prime overhang

9. The DNA Digestion Reaction
Restriction Buffer provides optimal conditions
• NaCI provides the correct ionic strength
• Tris-HCI provides the proper pH
• Mg2+ is an enzyme co-factor

10. Agarose Electrophoresis Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Buffer
Dyes
Agarose gel
Power Supply

11. Agarose Electrophoresis Running
• Agarose gel sieves DNA fragments according to size
– Small fragments move farther than large fragments
Gel running
Power Supply

12. Analysis of Stained Gel
Determine
restriction fragment
sizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
Identify the related
samples

13. Molecular Weight Determination
Fingerprinting Standard Curve: Semi-log
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,

14. Agarose Gel Electrophoresis
The standard method for separating DNA fragments is electrophoresis through agarose gels.

15. Agarose Gel Electrophoresis
The standard method for separating DNA fragments is electrophoresis through agarose gels.
Agarose is a polysaccharide like agar or pectin derived from seaweed

16. Agarose Gel Electrophoresis
The standard method for separating DNA fragments is electrophoresis through agarose gels.
Agarose is a polysaccharide like agar or pectin derived from seaweed
It dissolves in boiling water and then gels as it cools

17. Agarose Gel Electrophoresis
A comb is placed in the liquid agarose after it has been poured
Removing the comb from the hardened gel produces a series of wells used to load the DNA

18. Agarose Gel Electrophoresis
DNA is applied to a slab of gelled agarose

19. Agarose Gel Electrophoresis
DNA is applied to a slab of gelled agarose
The sample is loaded with a loading buffer—containing dyes and glycerol or sugar

20. Agarose Gel Electrophoresis
DNA is applied to a slab of gelled agarose
The sample is loaded with a loading buffer—containing dyes and glycerol or sugar
Electric current is applied across the gel

21. Agarose Gel Electrophoresis
DNA is negatively charged (due to PO4)

22. Agarose Gel Electrophoresis
DNA is negatively charged (due to PO4)
Migrates from the negative (black) electrode to the positive (red) electrode.

23. Agarose Gel Electrophoresis
Rate of migration of DNA through agarose depends on the size of DNA

24. Agarose Gel Electrophoresis
Rate of migration of DNA through agarose depends on the size of DNA
Smaller DNA fragments move more quickly

25. Agarose Gel Electrophoresis
Rate of migration of DNA through agarose depends on the size of DNA
Smaller DNA fragments move more quickly
Rate of migration is inversely proportional to the log10 of molecular weight

26. Agarose Gel Electrophoresis

27. Agarose Gel Electrophoresis
Concentration of agarose also affects migration

28. Agarose Gel Electrophoresis
Concentration of agarose also affects migration
Higher concentration of agarose, the more it retards the movement of all DNA fragments

29. Agarose Gel Electrophoresis
Concentration of agarose also affects migration
Higher concentration of agarose, the more it retards the movement of all DNA fragments
Small DNA fragments require higher concentrations of agarose/ Lg fragments low concentrations

30. Agarose Gel Electrophoresis
Agarose gels must be prepared and run in a buffer containing ions.

31. Agarose Gel Electrophoresis
Agarose gels must be prepared and run in a buffer containing ions.
Ions are charged particles (like those found in salt) and are necessary to carry a charge

32. Agarose Gel Electrophoresis
During electrophoresis water undergoes hydrolysis : H2O  H+ and OH-

33. Agarose Gel Electrophoresis
During electrophoresis water undergoes hydrolysis : H2O  H+ and OH-
The anode (+ /red) pole becomes alkaline because OH- will accumulate at this pole
The cathode (-/black) pole becomes acidic because H+ will accumulate at this pole

34. Agarose Gel Electrophoresis
Buffers prevent the pH from changing by reacting with the H+ or OH- products

35. Agarose Gel Electrophoresis
The buffer is either TBE or TAE
TBE is made with Tris/Boric Acid/EDTA
TAE is made with Tris/Acetic Acid/ EDTA

36. Agarose Gel Electrophoresis
The voltage applied to the gel affects how quickly the gel runs

37. Agarose Gel Electrophoresis
The voltage applied to the gel affects how quickly the gel runs
The higher the voltage, the more quickly the gel runs………But that often reduces the quality of the DNA separation

38. Agarose Gel Electrophoresis
The voltage applied to the gel affects how quickly the gel runs
The higher the voltage, the more quickly the gel runs………But that often reduces the quality of the DNA separation
>>>>>>>>>>It also generates heat which reduces the quality of the DNA separation

39. Agarose Gel Electrophoresis
To make DNA fragments visible after electrophoresis, the DNA must be stained

40. Agarose Gel Electrophoresis A gel stained with Methylene blue