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 8.3, 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.

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

Uses

TAE (Tris-acetate-EDTA) buffer is used as both a running buffer and in agarose gels.[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.

Preparation

TAE buffer is commonly prepared as a 50× stock solution for laboratory use. A 50× stock solution can be prepared by dissolving 242 g Tris base in water, adding 57.1 ml glacial acetic acid, and 100 ml of 500 mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 litre. This stock solution can be diluted 49:1 with water to make a 1× working solution. This 1× solution will contain 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.

No. Name Per 1 mole 50x solution 50× 1× solution 1X
1 Tris base 121.1 g/L 2 M 242.2 g/L 40 mM 4.844 g/L
2 acetic acid 57.1 ml/L 1 M 57.1 ml/L 20 mM 1.21 ml/L
3 EDTA disodium salt dihydrate 372.24 g/L 50 mM 18.612 g/L 1 mM 0.372 g/L

2 M = 2000 mM so 2000 mM /50 = 40 mM for 1×.
1M = 1000 mM so 1000 mM /50 = 20 mM for 1×.
50 mM /50 = 1 mM for 1×.

First of all, these ingredients should be dissolved in 500 ml, then made up to 1000 ml.
Note: EDTA will take more time to dissolve, so while dissolving EDTA use magnetic stirrer (few amounts of EDTA in 3 or 4 times).

A step-by-step recipe of the preparation method for 50× TAE buffer is available on protocols.io.[6]

See also

References

  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
  6. ↑ Behle, Anna; Pawlowski, Alice (6 April 2018). "Recipe for 50x TAE buffer". doi:10.17504/protocols.io.gtvbwn6. {{cite journal}}: Cite journal requires |journal= (help)
This article is issued from Wikipedia. The text is licensed under Creative Commons - Attribution - Sharealike. Additional terms may apply for the media files.