What is the purpose of the buffer in gel electrophoresis?
Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. There are a number of buffers used for electrophoresis. The most common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA. In molecular biology it is used in agarose electrophoresis typically for the separation of nucleic acids such as DNA and RNA. It is made up of Tris-acetate buffer, usually at pH 8.3, and EDTA, which sequesters divalent cations.
- TBE or Tris/Borate/EDTA, is a buffer solution containing a mixture of Tris base, boric acid and EDTA. As these ions are necessary co-factors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation.
- TAE (Tris-acetate-EDTA) buffer is used as both a running buffer and in agarose gel. Its use in denaturing gradient gel electrophoresis methods for broad-range mutation analysis has also been described. TAE has been used at various concentrations to study the mobility of DNA in solution with and without sodium chloride.
- You can dissolve the acid in water by following a few steps. Mix the EDTA in with about 80 mL of distilled water. Add the NaOH pellets, which should bring the pH of the water up to 8.0, the necessary level to dissolve EDTA. Mix the solution vigorously with the magnetic stirrer until the EDTA dissolves.
Make a concentrated (5x) stock solution of TBE by weighing 54 g Tris base (FW = 121.14) and 27.5 g boric acid (FW = 61.83) and dissolving both in approximately 900 milliliters of deionized water. Then add 20 milliliters of 0.5 M EDTA (pH 8.0) and adjust the solution to a final volume of 1 liter.
- TBE Buffer, 10X (pH 8.3), is used for polyacrylamide and agarose gel electrophoresis. This product is optimized for use in DNA applications. Composition: 890mM Tris-borate, 890mM boric acid, 20mM EDTA.
- Gel electrophoresis is a widely used technique in life science laboratories to separate macromolecules such as DNA, RNA, and proteins. Gel electrophoresis is usually performed in labs to analyze DNA, RNA, or protein samples from various sources.
- To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.
The buffer is needed to maintain the pH of the DNA solution at close to neutral level because if it can become acidic through electrolysis. The electrical currents caused by the electrodes can cause water molecules to dissociate and release H+ ions.
- Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. There are a number of buffers used for electrophoresis. The most common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).
- How are DNA fragments separated using gel electrophoresis? A solution of DNA molecules is placed in a gel. Because each DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules.
- The most commonly used stain for detecting DNA/RNA is ethidium bromide. Ethidium bromide is a DNA interchelator, inserting itself into the spaces between the base pairs of the double helix. Ethidium bromide possesses UV absorbance maxima at 300 and 360 nm.
Updated: 16th October 2019