TAE buffer

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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.[1] It is made up of Tris-acetate buffer, usually at pH 8.0, and EDTA, which sequesters divalent cations. TAE has a lower buffer capacity than TBE and can easily become exhausted, but linear, double stranded DNA runs faster in TAE.

Recently, Brody & Kern simplified electrophoretic buffers by substituting TBE and TAE buffers for a more efficient and inexpensive conductive media in gel systems.[2]

Uses[edit]

TAE (Tris-acetate-EDTA) buffer is used as both a running buffer and in agarose gel.[3] Its use in denaturing gradient gel electrophoresis methods for broad-range mutation analysis has also been described.[4] TAE has been used at various concentrations to study the mobility of DNA in solution with and without sodium chloride.[5] However, high concentrations of sodium chloride (and many other salts) in a DNA sample retard its mobility. This may lead to incorrect interpretations of the resulting DNA banding pattern.

Compared with TBE buffer, TAE buffer offers advantages in subsequent enzymatic applications for the DNA sample. For example, if a DNA sample is going to be used in a cloning experiment, the step that follows its running on an agarose gel is to ligate (covalently link) to a cloning vector (most likely a plasmid). A DNA sample from a TAE gel is suitable for this purpose, while DNA from a TBE gel is not, because borate in the TBE buffer is a strong inhibitor for many enzymes[citation needed].

Preparation[edit]

TAE buffer is commonly prepared as a 50X stock solution for laboratory use. A 50X stock solution can be prepared by dissolving 242g Tris base in water, adding 57.1mL glacial acetic acid, and 100mL of 500mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter. This stock solution can be diluted 50:1 with water to make a 1X working solution. This 1X solution will contain 40mM Tris, 20mM acetic acid, and 1mM EDTA.

References[edit]

  1. ^ Ogden, R.C., and Adams, D.A., (1987) Electrophoresis in agarose and acrylamide gels. Methods Enzymol., 152:, 61-87.
  2. ^ Brody, J.R., Kern, S.E. (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13. doi:10.1016/j.ab.2004.05.054 PMID 15351274 PDF
  3. ^ Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, volume 3, apendices B.11 and B.23 ISBN 0-87969-309-6
  4. ^ Hayes, V.M. et al., (1999) Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nucleic Acids Res., 27(20): e29. PMID 10497279
  5. ^ Stellwagen, E., and Stellwagen, N.C. (2002) The free solution mobility of DNA in Tris-acetate-EDTA buffers of different concentrations, with and without added NaCl. Electrophoresis, 23(12): 1935-1941. PMID 12116139

See also[edit]