Chemistry:TAE buffer
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
- ↑ Ogden, R.C., and Adams, D.A., (1987) Electrophoresis in agarose and acrylamide gels. Methods Enzymol., 152:, 61-87.
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ 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
- ↑ Behle, Anna; Pawlowski, Alice (6 April 2018). Recipe for 50x TAE buffer. doi:10.17504/protocols.io.gtvbwn6. https://www.protocols.io/view/recipe-for-50x-tae-buffer-gtvbwn6.
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