1. Classification
Gel electrophoresis is divided into vertical types (including column gels and slab gels) and horizontal types (mainly slab gels) (Figure 6-18). Generally, vertical separation is slightly superior to horizontal, but horizontal gel preparation has at least four advantages: there is support beneath the entire gel, allowing the use of low-concentration agarose; it is possible to prepare agarose gel plates of different specifications; gel preparation and sample loading are more convenient; the electrophoresis chamber is easy to construct and cost-effective. Since horizontal electrophoresis is performed with the agarose gel plate fully submerged about 1mm below the surface of the electrophoresis buffer, it is also called submerged electrophoresis.
Electrophoresis Tank DYCP-31DN for agarose gel electrophoresis
2.Buffer System
In nucleic acid separation, most systems adopt continuous systems. Commonly used electrophoresis buffers include TBE (0.08mol/L Tris·HCl, pH 8.5, 0.08mol/L boric acid, 0.0024mol/L EDTA) buffer and THE (0.04mol/L Tris·HCl, pH 7.8, 0.02mol/L sodium acetate, 0.0018mol/L EDTA) buffer. These buffers are generally prepared as 10x stock solutions and diluted to the required concentration when in use. The migration rates of linear and circular DNA in agarose gel vary with the buffer used. In THE buffer, the migration rate of linear DNA is greater than that of circular DNA, while in TBE buffer, the opposite is true.
3.Preparation of Agarose Gel
(1) Preparation of Horizontal Agarose Gel
(a) Prepare the required concentration of agarose gel using 1x electrophoresis buffer.
(b) Heat the agarose to complete dissolution either in a boiling water bath, on a magnetic stirrer, or in a microwave. Cool the agarose solution to 55°C and add ethidium bromide (EB) dye to a final concentration of 0.5 μg/ml.
(c) Seal the edges of glass or acrylic plates with a small amount of agarose gel, add a comb, and position the comb teeth about 0.5~1.0 mm above the plate.
(d) Pour the melted agarose gel solution continuously into the glass or acrylic plate mold (the thickness depends on the volume of the DNA sample), avoiding the introduction of air bubbles. Let it solidify naturally at room temperature.
(e) Carefully remove the comb after complete solidification. Add an appropriate amount of electrophoresis buffer to the gel tank, ensuring that the gel plate is submerged about 1 mm below the surface of the electrophoresis buffer.
(2) Preparation of Vertical Agarose Gel
(a) Remove grease or residue from glass plates by washing with ethanol.
(b) Place spacer plates between front and back dams, align the edges of the spacer plates with the front and back dams, and secure them with clamps.
(c) Add 2% agarose in 1x buffer between the edges of the spacer plates to form a 1 cm high agarose plug at the bottom of the gel casting chamber.
(d) Pour the melted agarose gel at the desired concentration, prepared in 1x buffer, into the gel chamber up to 1 cm below the top.
(e) Insert the comb, avoiding trapping air bubbles under the comb teeth. Sometimes, wrinkles may appear at the comb teeth during agarose gel cooling; in such cases, simply add some melted agarose at the top to solidify it.
(f) Remove the comb. To prevent buffer leakage in the loading slot, seal the connection between the agarose gel plate and the electrophoresis chamber with 2% agarose and add the required amount of buffer.
(g) Add 1x electrophoresis buffer to the gel chamber.
(h) Carefully load DNA samples onto the agarose gel beneath the buffer.
More information about the basic knowledge about agarose gel electrophoresis, we will share next week. Wish these information be helpful to your experiment.
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Post time: Dec-07-2023