CORE PRACTICAL FOUR: GEL ELECTROPHORESIS

DESCRIBE HOW GEL ELECTROPHORESIS CAN BE USED TO SEPERATE DNA FRAGMENTS LENGTH

INTRODUCTION

 Agarose gel electrophoresis is a powerful, analytical method for the separation of biomolecules.

 This experiment demonstrates the basic procedures of agarose gel electrophoresis, including gel
casting, sample application and separation using a selected set of tracking dye that have different
properties and various rates of mobility during electrophoresis.

 The purpose of the gel might be to look at the DNA to quantify it or to isolate a particular band.

 The DNA is visualised in the gel by addition of ethidium bromide. This binds strongly to DNA by
intercalating between the bases and it absorbs invisible UV light and transmits the energy as visible
orange light.

 Electrophoresis equipment is used to separate macromolecules, either nucleic acids or proteins, on

the basis of size, electric charge, and other physical properties.

PRINCIPLE

 Electrophoresis is a technique used to separate macromolecules especially proteins and nucleic acids
that differ in size and charge.

 When charged molecules are placed in an electric field, they migrate toward either the positive or
negative pole according to their charge. Nucleic acids have a consistent negative charge imparted
by their phosphate backbone, and migrate towards anode.

 Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5

to 2%. The higher the agarose concentration the stiffer the gel.

 By varying the concentration or agarose. fragments of DNA from about 200 to 50.000 bp can be
separated using standard electrophoretic techniques

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MATERIALS REQUIRED

Submarine Gel Electrophoresis Unit with power supply, Distilled water,

Micropipettes and tips, masking tape,

UV transilluminator, sample DNA,

Agarose, 10x TEB Buffer,

Gel loading dye, Ethidium bromide

PROCEDURE:

Steps Description
1 Wash the gel tray and comb(s). Tape the ends or the casting tray using masking tape

2 Place comb in the correct orientation across the end of the gel holder.

3 Mix agarose with 25 ml of O.5x TEB to the appropriate final concentration of

1% gel

4 When the gel temperature is around 400C, add 2µl or ethidium Bromide (final
concentration 0.5µpg / ml) and mix properly.

5 Seal the gel casting tray on both sides and place the comb on the gel tray in
appropriate place.
6 Pour the agarose mixture into the tray-containing comb.

7 After complete solidification of agarose

remove the seal from either sides of the tray without
disturbing the gel.

8 Keeps the gel tray in tank containing 0.5 x TEB buffer with the wells in the cathode
(negative) side. The buffer level is maintained above the gel tray.

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9 Gently lift the comb without damaging the wells, and now
the gel is ready for loading.

10 Connect the power cords between the electrophoresis tank and the power pack
before loading the samples.

11 Prepare samples for electrophoresis, by adding 2µl of gel loading dye into 5 µl of
given DNA sample and mix well by pipetting. Load the sample in the well.
12 After loading switch on the power pack and adjust the voltage
to 50v or 100v , Continue the electrophoresis until the dye
reaches to 1/3 rd of the gel or above

13 Observe the bands under UV by using UV Transilluminator

RESULT & INTERPRETATION:

PRECAUTIONS :

 Agarose can become superheated and violently boil over. Exercise caution when heating.
 Swirl flask occasionally during heating. Heat until close inspection reveals that the agarose is 100%
dissolved. Undissolved agarose will appear as little flecks.

Health Hazard

 Ethidium bromide is a powerful mutagen and is moderately toxic and should be handled with care.
 Wear gloves when handling contaminated equipment or solutions containing ethidium bromide
 Confine the compound to the restricted area.
 Use plastic wrap to protect equipment and surfaces from being contaminated. UV is also
carcinogenic and must not be allowed to shine on naked skin or eyes. So wear face protection, gloves
and long sleeves.

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SAQ1. (a) In the formation of recombinant DNA many different restriction endonuclease enzymes are used.
Each enzyme cuts the DNA of a plasmid at a specific base sequence called a restriction site.

The diagram shows the position of restriction sites, A, B, C and D, for each of four different enzymes on a
plasmid.

The distance between these sites is measured in kilobases of DNA.

The plasmid was cut using only two of the restriction enzymes. The resulting fragments were separated by gel
electrophoresis. The positions of the fragments are shown in the chart.

(i) Which of the restriction sites were cut? [1]

(ii) Explain your answer. [1]

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