Restriction Digestion and Analysis of Lambda DNA Kit

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

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

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

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 .

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

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

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

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

- + 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).

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

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.

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

• 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

+ - 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

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

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

DNA Marker Standard Curve Size (bp) Distance (mm) 23,000 11.0 9,400 13.0 6,500 15.0 4,400 18.0 2,300 23.0 2,000 24.0