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LAB PRACTICAL 4

TITLE: AGAROSE GEL ELECTROPHORESIS


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to determine the size and quality of the extracted DNA from Blood.

Introduction:
Electrophoresis is a technique used to separate ad sometimes purify macromolecules especially
proteins and nucleic acids that differ in size, charge or conformation. As such, it is one of the most
widely used technique in biochemistry and molecular biology. When charged molecules are placed in
an electric field, they migrate toward either the positive or negative pole according to their charge. In
contrast to proteins which can have either a net positive charge, nucleic acids have a consistent
negative charge imparted by their phosphate backbone and migrate toward the anode.
The most commonly technique for nucleic acid separation is agarose gel electrophoresis. Agarose gels
are ideal for RNA and DNA because agarose is inserted to these molecules and the molecules are
easily recovered from the gel for further use. Nucleic acids will separate by size in agarose without
the need for chemical modification. Agarose is easily prepared by dissolving agarose (refine from
sugar) in heated buffer solution.
Materials:

-agarose powder
10xTAE/TBE buffer
Ethium Bromide
Loading dye
1kb DNA ladder
Deionised distilled water
Gel chamber with gel tray and comb
100mL Erlenmeyer Flask.

Procedure:
1. Prepared 0.8% agarose gel by weighing out of the agarose powder and transfer into 100mL
Erlenmeyer Flask.
2. Added 50mL of 10x TAE/TBE buffer and swirl the flask to ensure homogenous distribution
of the agarose powder in the buffer.
3. The mixture is heated until it begins to boil. Swirl the flask very well to make sure all agarose
is dissolved in the buffer (no solid powder form should be visible).Continue until a clear
translucent solution is formed.
4. Set aside to cool down just enough to where the solution will not melt the gel tray.
5. While the gel solution is cooling, filled the gel chamber with 10xTAE/TBE.
6. Once the agarose buffer has cooled slightly, added 2L of ethidium bromide into the solution
and poured into the gel tray with well comb fixed properly.
7. After the gel has cooled completely and solidified the combs can be removed and the tray
inserted properly into the gel chamber.
8. To cover the gel, poured enough 10xTAE/TBE buffer into the chamber and filled the wells.
9. Into the wells, pipette 6L of 1Kb DNA ladder(mixed with Loading dye) and 12L of
samples (mixed with Loading dye)
10.Closed the lid of gel chamber.
11. For 30 minutes, run the gel at 100V.
12.Under the UV light, visualized the gel.

QUESTIONS:
1. State the function of ethidium bromide.
2. What is the factor that determines the percentage of agarose gel that will be used in
electrophoresis?
3. The application of gel electrophoresis.