LAB PRACTICAL 4

TITLE: AGAROSE GEL ELECTROPHORESIS

- 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 2µL 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 6µL of 1Kb DNA ladder(mixed with Loading dye) and 12µL 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.