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Electrophoresis
Electrophoresis is the movement of molecules by an electric current .This is practically done in a matrix to limit migration and contain the migrating material.
Electrophoresis is routinely applied to the analysis of proteins and nucleic acids
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Gel Systems
• Agarose Gels
• Polyacrylamide Gels
• Capillary Electrophoresis
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Gel electrophoresis have either a horizontal or vertical format
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Agarose Gels
• Agarose is a polysaccharide polymer extracted from seaweed.
• Agarose powder is dissolved in running buffer (either T.B.E or T.A.E).
• Agarose gels usually have a range of 0.5% (any lower and there is not enough tensile strength to hold together) up to about 4% (any high is too viscous to work with).
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Agarose Gels
• Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix.
• Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel
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• The concentration of the agarose dictates the size of the spaces in the gel.
• Small pieces of DNA (50–500 bp) are resolved on more concentrated agarose gels, e.g., 2%–3% .
• Larger fragments of DNA (2000–50,000)
are best resolved in lower agarose concentrations, e.g., 0.5%–1%.
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Resolution of double-stranded DNA fragments on 2%, 4%, and 5% agarose
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Procedure
• Prepare sufficient electrophoresis buffer (usually 1x TAE )
to fill the electrophoresis tank and to cast the gel
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• Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration.For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.
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• .
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• Heat in a microwave oven until the agarose dissolves. The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. wear gloves and hold it at arm's length
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• Use insulated gloves to transfer the flask/bottle into a water bath at 55°C.
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When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling.
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Pour the warm agarose solution into the mold.
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Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer
on the top of the gel, and carefully remove the comb.
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Pour enough 1X TAE electrophoresis buffer to cover the gel in the chamber
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Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.
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• Slowly load the sample mixture into the slots of the submerged gel using automatic micropipette. Load size standards into slots on both the right and left.
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• Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes).
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• Run the gel until the bromophenol blue and xylenecyanol
FF have migrated an appropriate distance through the gel.