Dna Electrophoresis Lab

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DNA Extraction from Wheat Germ and Making an Agarose Gel
AIM:
To be able to make and agarose gel and perform gel electrophoresis in six different dyes. Also, to extract DNA from wheat germ. INTRODUCTION:
Agarose gel is a substance that is used in science for gel electrophoresis and size exclusion chromatography. These processes use agarose gel to separate and analyze proteins and DNA. The medium is composed of a purified agarose powder that has been boiled in a buffer solution and then cooled into a gel. Agarose gel is most commonly associated with gel electrophoresis. In this procedure, scientists use an electrical charge to move deoxyribonucleic acid, more commonly known as DNA, or ribonucleic acid (RNA) through a gel matrix toward a positive pole. Because the molecules have to move through small holes in the lattice bonds in the agarose gel, smaller molecules move much faster than larger molecules. Using ultraviolet imaging of the molecules' movement and a formula that relates molecular weight to the speed of travel through the gel, scientists can determine the size of the molecules.

Electrophoresis is essentially a sieving process. The larger the fragment of DNA, the more easily will it become entangled in the matrix and, therefore, the more slowly will it migrate. Small fragments, therefore, run more quickly than large fragments at a rate proportional to their size. The relationship of size to migration rate is linear throughout most of the gel, except for the very largest fragments. Large fragments have a more difficult time penetrating the gel and their migration, therefore, does not have a linear relationship to size. The matrix can be adjusted, though, by increasing the concentration of agarose (tighter matrix) or by decreasing it (looser matrix). A standard 1% agarose gel can resolve DNA from 0.2 - 30 kb in length.

Agarose is extracted in the form of agar from several species of red marine algae, or seaweed, found in California and eastern Asia. Agar, a term derived from the Malay word agar-agar, meaning jelly, is typically derived from the Gelidium genera of seaweed. It is composed of both agarose and agaropectin molecules and provides support to the cell walls within the marine algae. Removed from the plant, the agarcan be used as a food thickener, much like gelatine, a laxative, or a medium for growing bacteria, fungus, or other microorganisms, when purified.

MATERIALS:
Part 1 - Making an Agarose Gel
small 8cmx10cm gels (minigels)
30-50mL of agarose (0.5g agarose and 60mL of 1xTBE)
20 microlitres restriction digest
5 microlitres of loading buffer
a comb
250mL conical flask
microwave
stirring rod
distilled water

Part 2 - Principles and Practice of Agarose Gel Electrophoresis •pre-made agarose gel
6 different coloured dyes
micropipette
direct current (D.C.) power source
white background
Part 3 - DNA Extraction from Wheat Germ
raw wheat germ
liquid detergent
50 - 60ºC tap water
alcohol (not rubbing alcohol) 14mL
50mL test tube
graduated cylinder (2)
wooden applicator stick
eyedropper
sealable container
hot plate
paper towels

PROCEDURE:
Part 1 - Making an Agarose Gel
0.5grams of agarose was added to a 250mL conical flask
60mL of 1xTBE was then added to the flask as well
The flask was swirled to mix
The mixture was placed in the microwave for 20seconds, then left to cool for 10minutes

Part 2 - Principles and Practice of Agarose Gel Electrophoresis Pre-made agarose gel was poured into a small gel tank 8cmx10cm The end blocks were removed and the comb was then submerged into the gel It was left to stand for 30minutes

The tray was flipped around so that the cathode and anode could match to its respective position (e.g. red match with red etc) Each person in the group got a chance to fill the well in the gel with a different colour dye After all wells...
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