Tris and EDTA are two major components which are used throughout the DNA extraction protocol. Tris and EDTA are applicable in lysis buffer preparation, elution buffer preparation and washing buffer preparation.
The major role of TE buffer in DNA extraction is to dissolve DNA into liquid form. However, it is also used as a lysis buffer.
In this present article, we will discuss the structure and function of Tris and EDTA and their role in DNA extraction.
- Importance of TE buffer
- How to prepare 10X TE buffer
- How to prepare 1X TE buffer
Tris is a (hydroxymethyl)aminomethane with the molecular formula (HOCH2)3CNH2. The major role of the Tris in any biological practice is to maintain the pH of the solution.
Majorly, Tris is used in biochemistry, molecular biology and chemistry. The pKa value of Tris is 8.07 (at 25°C).
Furthermore, It can also increase the permeability of the cell wall. Tris is available in powder form and dissolved between the pH 7.0 and 9.0. The structure of Tris is shown in the figure below,
EDTA, Ethylenediamine tetra-acetic acid (EDTA) has wide application in medicines, industries, laboratories and in cosmetics.
It is a colourless (white as in powder form), water soluble and organic molecule. However, it can be dissolved in water at high pH (pH nearby 8.0).
The chemical formula of EDTA is C10H16N2O8. The structure of EDTA is shown in the figure below. The EDTA works as a chelating agent in the DNA extraction.
It chelates the metal ion present into the enzymes and as we all know that the metal ions are the cofactor which increases the activity of the enzyme.
By chelating the metal ions, it deactivates the enzyme, therefore, reduces the activity of DNase and RNase.
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Importance of Tris EDTA (TE) buffer in DNA extraction
It dissolves DNA or RNA and protects the nucleic acid from degradation.
It is a major constituent of DNA extraction buffer which helps in lysis of cell wall and nuclear membrane.
It protects the nucleic acid from degrading by DNase or RNase.
It is likewise practised into the preparation of TBE and TAE buffer. TAE and TBE buffer are mainly involved in agarose gel electrophoresis of DNA. Here the duo maintains the constant pH environment into the solution while electrophoresis.
Further, TE buffer used in reviving the DNA primers.
TE buffer is a DNA preservative which stores DNA in intact form for a longer period of time, without degrading it.
How to preparation 10X TE buffer
Preparation of 10X TE buffer for 100ml of solution
- 100mM Tris HCl: 1.57gm
- 10mM EDTA: 0.292 gm
- Weigh 1.57 gm of Tris powder and 0.3722gm of EDTA into the flask.
- We have to add 98.1 ml of D/W but we also have to adjust pH with HCl hence add only half amount of D/W and adjust the pH of the solution with HCl until the pH reaches nearby 8.0.
- Now add the D/W to make the final volume of 100ml.
- In the preparation of TE buffer, pH plays a crucial role in performing a proper function.
How to prepare 1X TE buffer
- Our stock solution is 10X which means the total concentration of the stock solution is 10 times more than the working solution.
- Hence for preparation of 1X TE buffer, we have to take 1 ml of stock buffer and add 9ml of D/W.
- This will make a 1X working solution from a 10X stock solution.
- 10mM Tris HCL and 1mM EDTA will make directly the 1X of working solution.
- In order to achieve a good quality of results please autoclave the TE buffer solution before use.
Alternatively, the DNA can also be dissolved in water. However, the chance of acid hydrolysis is high in water (because water is slightly acidic) which can be prevented by storing DNA into the TE buffer.
So the TE buffer is the best nucleic acid storing solution apart from all other solutions.
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Significantly, the Tris and EDTA are two important chemicals used in DNA extraction as a constituent of extraction buffer, elution buffer and as a storage buffer. I have personally used TE as an extraction buffer and believe me, Tris- EDTA buffer worked excellently with proteinase K DNA extraction method.
Water is the alternative option for dissolving DNA. However, it can not be used for storing DNA for long-term use. still, if you want to dissolve DN into water use MiliQ water or DD/W. Remember, do not use tap water.
Tips: For a short-term or single-use, dissolve DNA in water, because of the slight acidity of water the DNA dissolves fast and easily.