1. Restriction Digestion and Analysis of Lambda DNA Kit

2. Why electrophoresis? To separate DNA fragments from each other
To determine the sizes of DNA fragments
To determine the presence or amount of DNA
To analyze restriction digestion products

3. What can you do with the Restriction Digestion and Analysis of Lambda DNA Kit?
Understand the use of restriction enzymes as biotechnology tools and the mechanics of a restriction enzyme digest
Become familiar with principals and techniques of agarose gel electrophoresis
Estimate DNA fragment sizes from agarose gel data
Understand the importance of restriction enzymes and their applications

4. What are restriction enzymes?
Used by bacteria to protect against viral DNA infection
1950s: discovery of primitive immune system in bacteria
1962: proof that in bacteria, an enzyme system recognized and destroyed foreign DNA while protecting its own
1960s: E.coli extracts isolated which cleaved phage DNA, but not useful
1970s: H. influenzae, HindII, extract isolated with no modification activity and cleavage within the restriction site
Endonucleases = cleave within DNA strands
Exonucleases = digest from the ends of DNA molecules
Types I, II, and III
3,139 known enzymes

5. Introduction to Agarose Gel Electrophoresis
Agarose gel electrophoresis is a method to separate DNA or RNA molecules by size.
This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).
Shorter molecules move faster and migrate faster than longer ones .

6. Principle of electrophoresis
powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes
convenient analytical method for determining the size of DNA molecules in the range of 500 to 30,000 base pairs.
employs electromotive force to move molecules through a porous gel

7. Principle (cont.) separates molecules from each other on the basis of
size and/or
charge and/or
shape
basis of separation depends on how the sample and gel are prepared

8. Material required for agarose gel electrophoresis
Electrophoresis chamber
Agarose gel
Gel casting tray
Buffer
Staining agent (dye)
A comb
DNA ladder
Sample to be separate

9. Bio-Rad’s Electrophoresis Equipment
Power Supplies
Precast Ready
Agarose Gel

10. - + Cathode Anode Buffer Dyes Agarose gel Power Supply
During electrophoresis, water is electrolyzed which generates protons (H+ ions)at the anode (positive) and hydroxyl ions (OH -1)at the cathode (negative). The cathode (negative) end of the electrophoresis chamber then becomes basic and the anode (positive) end becomes acidic.
The electrode at which electrons enter the gel box from the power supply (along the black wire) is called the cathode and is negative (-). The electrode at which electrons leave the box and re-enter the power supply (along the red wire) is called the anode and carries a positive charge (+). The flow of electrons sets up a potential energy difference between the electrodes. This is known as potential, and is measured in volts. It establishes an electric field through which the ions in the gel box fluid migrate. The migration of ions in the fluid creates electrical current which is measured in milliamperes (milliamps).

11. What is Agarose ?
A linear carbohydrate polymer extracted from seaweed , agarobiose
forms a porous matrix as it gels
shifts from random coil in solution to structure in which chains are bundled into double helices

12. Applied voltage  voltage,  rate of migration
The higher the voltage, the more quickly the gel runs
But if voltage is too high, gel melts
The best separation will apply voltage at no more than 5V/cm of gel length.

13. Staining of DNA
To make DNA fragments visible after electrophoresis, the DNA must be stained
The favorite—ethidium bromide
When bound to DNA it fluoresces under ultraviolet light (reddish –orange colour)
Convenient because it can be added directly to the gel
Sensitive—detects 0.1ug of DNA

14. • DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2
• An agarose gel is used to slow the movement of DNA and separate by size. + -
Power

15. + - How fast will the DNA migrate? Power
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA + -
Power
small
large

16. Actual Results of Restriction Enzyme Digestion
Lane 1, DNA markers (HindIII lambda digest)
lane 2, uncut lambda DNA
lane 3, lambda DNA digested with PstI
lane 4, lambda DNA digested with EcoRI
lane 5, lambda DNA digested with HindIII

17. Analysis of DNA Fragments
Determine restriction fragment sizes
Create standard curve using DNA marker
Measure distance traveled by restriction fragments
Determine size of DNA fragments

18. DNA Marker Standard Curve
Size (bp) Distance (mm)
23, 9, 6, 4, 2, 2,