Selection and operation precautions of three Tris buffers for agarose gel electrophoresis

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The agarose gel has a network structure, and the substance molecules are subjected to resistance when passing through this network, and the resistance of macromolecular substances is greater when they are surging. In gel electrophoresis, the separation of charged particles depends not only on the net charge The nature and quantity also depend on the size of the molecule, which will increase the resolution of the DNA band. There are several agarose gel electrophoresis buffers suitable for the electrophoresis of natural double-stranded DNA. The choice of electrophoresis buffer has a certain influence on the electrophoresis results. The use of a new buffer during electrophoresis can significantly improve the electrophoresis effect. The three commonly used electrophoresis buffers are all Tris salt solutions: TAE (Tris-acetate), TBE ( Tris-phosphate), TBE* (Tris-borate).




Preparation of three agarose gel electrophoresis buffers:

These three electrophoresis buffers are suitable for electrophoresis, but there are some differences:

1. TAE has the lowest buffer capacity and will be consumed for a long time. At this time, the positive side of the gel will be acidified, and the color of bromophenol blue migrating to the anode in the gel will change from blue purple to yellow, and the buffer solution should be changed regularly.

2. TBE concentrated storage solution is easy to form a precipitate after long-term storage. To avoid this problem, the 5X solution can be stored in a glass bottle at room temperature, and it should be discarded after precipitation. The lXTBE used for polyacrylamide gel electrophoresis is twice the concentration of the solution used for agarose gel electrophoresis. The buffer tank of the vertical tank of polyacrylamide gel is small, and the amount of current passing through the buffer is usually large. It is necessary to use 1XTBE to provide sufficient buffer capacity.

3. TAE buffer is usually replaced 2 to 3 times, and TBE buffer can be used about 10 times.

4. Alkaline agarose gel electrophoresis buffer should be used now. When preparing agarose gel electrophoresis buffer, three kinds of EDTA need to be added in order to remove divalent metal ions. Boric acid and agarose easily form a complex, which makes the agarose gel negatively charged, which causes adsorption of positively charged substances.

5. TBE is usually prepared and stored as a 5× or 10× stock solution. The pH of the stock solution at this concentration is about 8.3, and it needs to be diluted before application, and the gel solution and agarose gel electrophoresis buffer should be prepared with the same stock solution.

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