Agarose Gel Electrophoresis

PRESENTED BY:-

AVINASH SHARMA

PALB-3235

power supply

gel box

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Contents:-

Definition of agarose gel electophoresis

Procedure of agarose gel electrophoresis

Application of gel electrophoresis

Problem in agarose gel electrophoresis

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What is agarose?• it is a polymer of D,L galactose and it is a heteropolysaccharide extracted

from red algae seaweed.

• Low electro endosmosis and good for working.• Agarose has large range of separation but low resolving power.

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Agarose Gel ElectrophoresisGel electrophoresis is a procedure that separates molecules on the basis of size and/or charge and/or shape under the influence of an electrical field. It is based on the principle of “ohms law equation” and”power equation”.

Gel electrophoresis is a widely used technique for the separation of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the separation of DNA.

e will be using agarose gel electrophoresis to determine the presence and size of PCR products. + -

Power

DNAHO2

+

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Procedures of agarose gel electrophoresis:-

An agarose gel is prepared by combining agarose powder and a buffer solution.

Agarose�

Buffer�

Flask for boiling�

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Casting trayGel combs

Power supply

Gel tank Cover

Electrical leads

Electrophoresis Equipment

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Gel casting tray & combs

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Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.

Preparing the Casting Tray

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Agarose Buffer Solution

Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.

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Agarose is insoluble at room temperature (left).The agarose solution is boiled until clear (right).

Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.

Melting the Agarose

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Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles.

Pouring the gel

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Each of the gel combs should be submerged in the melted agarose solution.

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When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape. 26/04/14

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Place the gel in the electrophoresis chamber.

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buffer

Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

Cathode(negative)

Anode(positive)

wells

DNA

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Loading Buffer: • Bromophenol Blue (for color) • Glycerol (for weight)

Sample Preparation

Mix the samples of DNA with the sample loading buffer ( tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells.

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Loading the Gel

Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

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Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Running the Gel

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wells Bromophenol Blue

Cathode(-)

Anode(+)

Gel

After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA(-)

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100 200 300

1,650

1,000

500

850

650

400

12,000 bp

5,000

2,000

DNA Ladder Standard

Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

-

+

DNAmigration

bromophenol blue

Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule

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Staining the Gel

***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

• Ethidium bromide fluorescent dye and binds to the DNA under UV light allowing the visualization of DNA on a Gel.

• Ethidium bromide can be added to the gel .

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Staining the Gel

• Place the gel in the staining tray containing warm diluted stain.• Allow the gel to stain for 25-30 minutes.• To remove excess stain, allow the gel to destain in water.• Replace water several times for efficient destain.

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Ethidium Bromide requires an ultraviolet light source to visualize

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Factors affecting the DNA in gel elctrophoresis:-

• Agarose concentration:-↑ conc. Of agarose then migration larger fragments of dna and vice versa so for

larger fragments of dna agarose conc.=0.7% and smaller fragments of dna agarose conc.=1.5%.• Voltage:-Voltage increases then migration of larger fragments of dna will be more than

the smaller dna.so voltage should not be more than 5volts/cm.• Electrophoresis buffer:-TAE and TBE buffers are used in the gel electrophoresis because it establish

the pH8.0 and provide ions for the migration of dna and ionic strength are different.• Effects of ethidium bromide:-It is a carcinogenic mutagen and fluorescent dye which are used in the

visualizing the dna when agarose gel are placed in the UV transilluminator then dye are free in the gel then dye binds with dna then it produce orange light for visualizing the dna.

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Application of electrophoresis

• To separate fragments from each other• To determine the sizes of fragments• To determine the presence or amount of DNA• To analyze restriction digestion products prior to southern transfer or

of RNA prior to northern transfer.• Analysis of PCR product.

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Proplems in agarose gel electrophoresis:-• Smearing

•voltage too high for large fragments•too much DNA

• Gel melts•voltage too high•ionic strength too low

• Poor resolution•wrong [agarose]•small bands are fuzzy – the gel run may have been too long at too low a voltage, allowing diffusion of the DNA and broadening of the band

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Thank you for your attention