The aim of this experiment is to investigate the process of Electrophoresis and successfully analyse DNA fragments. Hypothesis:
That the experiment will show the visual representation of DNA proteins, and that the shorter the band the further is will travel. Background:
Restriction enzymes are DNA-cutting enzymes found in bacteria, which cut DNA into smaller fragments. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides, known as restriction sites. Restriction enzymes recognize a specific sequence of nucleotides and produce a double-stranded cut in the DNA. The recognition sequences usually vary between 4 and 8 nucleotides, and many of them are palindromic, meaning the base sequence reads the same backwards and forwards. Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose gel. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel. Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel, and identified using a DNA protein ladder. A restriction map is a map of known restriction sites within a sequence of DNA. Restriction mapping requires the use of restriction enzymes. A gel pattern is used to examine DNA bands, where where the molecules in the gel can be stained to make them visible. After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation. The ethidium bromide fluoresces reddish-orange in the presence of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white.
Figure 1: Gel Electrophoresis diagram
Figure 2: Electrophoresis Tank and view of experiment
Goggles Incubator Apron Agarose Gel
Gloves Gel tray and comb
Micropipettes Electrophoresis Tank
Minicentrifuge Power supply for tank
Vortex λ Bacteriophage DNA
Disposable tips Tip Tray Eco RI (restriction enzyme) Water filled container for used tips 10x buffer (EcoRI) BamHI (restriction enzyme) 10x buffer (BamHI) HindIII (restriction enzyme) 10x buffer (R+) Distilled water TAE Buffer solution
Staining card UV light viewing chamber
Day 1: DNA Digestion
1.Equipment gathered and safety procedures followed.
2.Eppendorf Tubes were labelled 1-4.