electrophoresis electrophoresis is the movement of molecules by an electric current.this is...

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

Gel Systems

• Agarose Gels

• Polyacrylamide Gels

• Capillary Electrophoresis

Gel electrophoresis have either a horizontal or vertical format

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).

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

• 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%.

Resolution of double-stranded DNA fragments on 2%, 4%, and 5% agarose

Procedure

• Prepare sufficient electrophoresis buffer (usually 1x TAE )

to fill the electrophoresis tank and to cast the gel

• 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.

• .

• 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

• Use insulated gloves to transfer the flask/bottle into a water bath at 55°C.

When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling.

Pour the warm agarose solution into the mold.

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.

Pour enough 1X TAE electrophoresis buffer to cover the gel in the chamber

Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.

• 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.

• 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).

• Run the gel until the bromophenol blue and xylenecyanol

FF have migrated an appropriate distance through the gel.