Buffers for Molecular Biology

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Why Tris Is Often Used in Genetic Engineering?

Chemical reactions that occur in molecular biology experiments generally proceed well only in a narrow range of pH values near neutrality, and many of the reactions generate or consume protons. For these reasons experiments are conducted in buffer solutions. The buffer solutions must also have the following properties:
  - Inert toward many compounds and enzymes
  - Highly polar
  - Safe and non-toxic
  - Inexpensive and easily available
  - Not affected by salt or temperature
  - No absorption of visible or ultraviolet light

There is no ideal buffer that has all of these properties. Buffers based on inorganic salts such as boric acid, bicarbonate, phosphoric acid, and ammonium salts have been used, but they can be used only in certain types of reaction systems and are not all-round.

In 1946, Gomori reported that organic amines can control pH in the range 6.5-9.7. Tris (Tris(hydroxymethyl)aminomethane or 2-Amino-2-hydroxymethyl-1,3-propanediol) was one of these amines. Since Tris buffer has properties suitable for many biochemical reactions, it has become one of the most used buffers in molecular biology experiments today. However, Tris also has some disadvantages. Its dissociation constant is affected by concentration and temperature; it is toxic to mammalian cells; and being a primary amine, it cannot be used together with DEPC (diethylpyrocarbonate). Therefore, caution should be exercised when it is used.

Applications of Buffers for Genetic Engineering

Buffer	Applications
Tris-HCl	General purpose buffer for genetic engineering experiments. pH adjustment is performed using hydrochloric acid. Stock solutions are prepared at a concentration of 1 M. The pH changes with temperature (a 1°C increase in temperature results in a 0.03 decrease in pH).
TE (Tris-EDTA)	TE buffer consists of Tris-HCl and EDTA. It is used to dissolve and preserve DNA, because EDTA chelates divalent metal ions necessary for the activity of nucleolytic enzymes and other enzymes.
Sodium Acetate	This is a buffer consisting of sodium acetate. A stock solution is prepared at a concentration of 3 M. It is used to neutralize the negative charge of nucleic acids to facilitate precipitation with ethanol.
Ammonium Acetate	This is a buffer consisting of ammonium acetate. A stock solution is prepared at a concentration of 10 M. It is used to neutralize the negative charge of nucleic acids to facilitate precipitation with ethanol.
TAE (Tris-acetate EDTA)	This buffer consists of Tris, acetic acid, and EDTA. A stock solution is prepared at a 50x concentration. It is mainly used for electrophoresis of nucleic acids. It is less expensive than the TBE buffer, provides a better separation of long DNA fragments, and provides a higher electrophoretic speed. On the other hand, its buffering capacity is inferior compared to the TBE buffer and it is not suitable for electrophoresis that requires a long running time.
TBE (Tris-borate EDTA)	This buffer consists of Tris, boric acid, and EDTA. A stock solution is prepared at a 10x concentration. It is mainly used for electrophoresis. It provides a better separation of short DNA fragments than the TAE buffer and is suitable for electrophoresis that requires a long running time.
MOPS	This is an aqueous solution of MOPS (3-(N-Morpholino) propanesulfonic acid). It is one of the Good’s buffers. pH adjustment is performed using potassium hydroxide. With added formaldehyde, it is used for denaturing electrophoresis of nucleic acids.
SSC (Saline Sodium Citrate)	This buffer consists of sodium citrate and sodium chloride. A stock solution is prepared at a 20x concentration. It is used for transfer and hybridization for Southern and northern blotting.

参考文献

1. Tamura, T.: “Bioreagent Preparation Pocket Manual”, Yodosha, Japan, (2004). (Japanese)
2. Green, R. M. and Sambrook, J.: ”Molecular Cloning A Laboratory Manual, 4th ed.”, Cold Spring Harbor Laboratory Press, United States, (2012).