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Preparation of Buffer stocks (TBE,TE and TAE)
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Objective



To study the properties of a buffer solution.


Theory

 

pH, the negative log of H+ ion concentration is an important factor in most of the biological reactions. The change in pH value can be resisted by adding suitable buffers to the reaction mixture. Gel electrophoresis, the basic technique in molecular biology experiment, requires buffer solutions for its effective working. In electrophoresis, the gel matrix through which the DNA samples will move is immersed in a buffer solution which will allows for electrical conductance and maintains a consistent pH. Buffers with higher concentration will allow the DNA molecules to migrate faster through the gel matrix. The buffers that are commonly used in gel electrophoresis are, Tris Acetate-EDTA (TAE) and Tris Borate-EDTA (TBE). TAE Buffer is used effectively for separating fragments which are larger than 4000bp and is also used to separate super coiled DNA. Whereas TBE Buffer is effective for the separation of fragments between 1 and 3000bp in length. It provides an ionic solution in order to allow the current to pass through the water.


X- Factor


It is better to prepare concentrated stocks of buffer solutions in order to save time and space. These concentrated stocks will last for a long period of time and can be easily diluted for use. These stocks are commonly labeled as X factors such as 10X, 5X, 100X etc. X-factor indicates that the solution is concentrated and must be diluted usually with water to 1X concentration for use.
For eg: - A 100X concentrated solution should be diluted to 100 fold.

 


Requirements of Biological Buffers


Solubility:
- A buffer solution should be fully soluble in water and sparingly soluble in other solvents. It is better to prepare concentrated stock solutions of these buffers such as 10X, 5X, or 100X at higher water solubility.


Permeability:
- A buffer should not be permeable through biological membrane. If do so, it will change the required concentration in the cell or organelles but this buffer has high degree of fat solubility and there by permeate membrane. So it becomes toxic for most of the mammalian cells.


Ionic strength: - It is necessary to maintain physiological ionic strength of a system as it is an important factor for most of the enzymatic reactions. A change in normal ionic strength may affect the catalytic activity of enzyme.


Complex formation:
- Complexes formed by buffer in the system should be soluble. Insoluble buffer complexes formed with metal ions may decrease the pH value by releasing protons.


Inert substances:
- A buffer should be inert, i.e. it does not subject to any enzymatic or non enzymatic changes.

 

Principle


In a buffer system, when the concentration of proton donor and its conjugate proton acceptor is equal, the addition of even small amount of Acid or alkali does not make any detectable change in the pH. This point at which there is no net charge is called as isoelectric point. The pH at isoelectric point is equal to pKa.


                                 [CH3COO¯ ]
pH = pKa + log   _______________
                                 [CH3COOH]

At isoelectric point [CH3COO¯] = [CH3COOH] hence, pH = pKa
Buffer capacity is the ability of a buffer solution to resist the change in pH when adding acid or alkali. Buffers with higher concentrations show higher buffering capacity.

 

 

 

 

 

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