TBE, also known as Tris Borate EDTA, is a buffer solution composed of Tris base, boric acid, and EDTA. It is commonly used in nucleic acid electrophoresis. This solution works effectively under somewhat basic conditions, preserving DNA deprotonated, water-soluble, and protected from deterioration. This concentration can be easily diluted to 1x or 0.5x before usage (with molecular biology-grade water).
TBE and TAE (Tris Acetate EDTA) buffers are often employed in molecular biology techniques involving nucleic acids, the most popular of which is electrophoresis. Tris-acid solutions are good buffers for somewhat basic environments because they retain DNA deprotonated and soluble in water. EDTA is a divalent cation chelator, notably magnesium (Mg2+). Because these ions are required cofactors for many enzymes, including contaminant nucleases, EDTA’s job is to shield the nucleic acids against enzymatic breakdown.
Tris has an effective range of 7-9. Boric acid has an effective pH range of 8-10. Based on this data, the overlap of buffering properties of both is 8-9; consequently, pH 8.3 was determined to be ideal. To make adjustments, NaOH/HCl is utilized.
Buffer preparation without adjustment is repeatable. Keep in mind that uncalibrated pH meters have some inaccuracy, thus it is occasionally advisable to use non-adjusted buffers if the pH of dissolved compounds is close to the predicted value.
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Reagents Required to Prepare TBE (Tris Borate EDTA) Buffer
- Boric acid
- Tris (tris(hydroxymethyl)aminomethane)
- Na2EDTA
- Distilled water
- HCl / NaOH (as per the requirements for adjustment)
Instruments Required to Prepare TBE (Tris Borate EDTA) Buffer
- Weighing balance
- Glass beaker
- Magnetic stirrer and pellet
- Pipette
- pH meter
- Measuring cylinders
- Autoclave or filter
Procedure For the Preparation of TBE (Tris Borate EDTA) Buffer
- Fill the 3/4th of the intended volume of distilled water into the beaker or flask.
- Using a weighing scale, prepare the appropriate quantities of chemicals. If you have an EDTA solution, measure the volume and mix it with water swiftly.
- The reagents should be dissolved in water. The pH should rise as the Tris base dissolves. The greater the pH, the easier it becomes for EDTA to dissolve (if using crystalline EDTA), although it is still a sluggish process. Particularly at this point, the magnetic stirrer comes in very handy.
- If necessary, adjust the pH using HCl.
- Add the distilled water to the obtained volume to reach the required volume.
- The solution should be autoclaved or filtered (particularly if you want to use it with RNA).
Preparation of 10x TBE (Tris Borate EDTA) Buffer
Reagents Required
- 108 g of Tris
- 55 g of Boric acid
- 40 ml of 0.5 M Na2EDTA (pH 8.0) / alternatively can use 9.3 g Na2EDTA
- 900 ml of distilled water
Procedure for Preparing 10x TBE (Tris Borate EDTA) Buffer
- In a flask/beaker keep 900 ml of distilled water, add 108 g of Tris and 55 g of Boric acid, and stir it well with a magnetic stirrer.
- Add 40 ml of 0.5 M Na2EDTA (pH 8.0) (or 9.3 g Na2EDTA) to the solution.
- Adjust the volume to 1 liter using distilled water.
- It can be stored at room temperature.
Preparation of 1x TBE (Tris Borate EDTA) Buffer
Reagents Required
- 10.8 g of Tris
- 5.5 g of Boric acid
- 4 ml of 0.5 M Na2EDTA (pH 8.0)
- 900 ml of distilled water
Procedure for Preparing 1x TBE (Tris Borate EDTA) Buffer
- In a flask/beaker keep 900 ml of distilled water, add 10.8 g of Tris and 5.5 g of Boric acid, and stir it well with a magnetic stirrer.
- Add 4 ml of 0.5 M Na2EDTA (pH 8.0) to the solution.
- Adjust the volume to 1 liter using distilled water.
- It can be stored at room temperature.
Applications
- TBE running buffer is the most often used buffer for DNA and RNA polyacrylamide gel electrophoresis.
- Tris-borate-EDTA buffer has been employed for pulsed-field gel electrophoresis (PFGE).
- Although TBE can be used for agarose gels, it is not advised in preparative gels used for nucleic acid recovery.
- TBE is utilized with non-denaturing or denaturing (7-M urea) gels. It is also commonly used for DNA automated sequencing gel.
Storage
- TBE (Tris Borate EDTA) Buffer can be stored at room temperature for up to three months (refrigeration may cause precipitation).
Caution
- Boric acid is poisonous. As a result, use gloves and avoid contact with it.
- Tris base is irritating to the skin and eyes.
- EDTA causes eye irritation.
Video Reference
Reference
- https://www.thermofisher.com/order/catalog/product/B52
- https://www.sigmaaldrich.com/NP/en/product/sigma/t4415
- https://sharebiology.com/tbe-buffer-tris-borate-edta-buffer/
- https://openwetware.org/wiki/TBE
