OVERVIEW

Here we describe the process of preparing a 5x TBE buffer. A 5x stock solution can be prepared by mixing and dissolving 54 gram Tris base, 27.5 gram Boric acid, and 20 ml of 0.5 M EDTA in water to a final volume of 1000 ml (cshlp-pdb.rec8458). The solution is sterilized by autoclaving and is stored at room temperature. 

REQUIREMENTS

Reagents and solutions
Tris base (C₄H₁₁NO₃, Molecular Weight: 121.14)
Boric acid (H₃BO₃, Molecular Weight: 61.83)
0.5 M EDTA stock solution (pH 8.0)
Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

COMPOSITION

Composition of 5X TBE electrophoresis buffer
0.445 M Tris borate
0.01 M EDTA
pH 8.2 – 8.4 (at 25°C)

Composition of 1X TBE electrophoresis buffer
89 mM Tris borate
2 mM EDTA
pH 8.2 – 8.4 (at 25°C)

OBJECTIVE

Preparation of 1000 ml of 5X TBE electrophoresis buffer, pH 8.3.

PROCEDURE

Step 1: To prepare 1000 ml of 5x TBE buffer, weigh out 54 g Tris base and 27.5 g boric acid. Transfer them to a 2 L beaker / conical flask. Add 800 ml deionized / Milli-Q water. Dissolve all ingredients by  mixing vigorously using a magnetic stirrer.

Note
You can also use acid-free EDTA powder to prepare a TBE buffer which is a better choice than using 0.5M EDTA solution (Sanderson et al., 2014). Although it takes some time to dissolve acid-free EDTA powder, the prepared solution serves as a better electrophoresis buffer by reducing the heat generation (Sanderson et al., 2014).

Tip
One can use manual shaking using a glass pipette to mix the ingredients. The magnetic stirrer makes the dissolving process automated and convenient.

Step 2: Add 20 ml of 0.5 M EDTA solution. Mix the solution again. Adjust the pH 8.3 if required.

Precaution
Since pH is dependent on temperature, we recommend adjusting the solution pH at room temperature (25°C).

Step 3: Adjust the solution volume to 1000 ml with deionized / Milli-Q water. Mix the solution again.

Step 4 (optional): One can filter the solution to remove any undissolved materials.

Step 5: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi), 121-124°C on liquid cycle).

Tips:
1. Transfer the solution to autoclavable bottles. There should be sufficient empty space ≈ 15 – 20%) in the autoclave bottle after filling the solution. Don’t forget to loosen the cap slightly.
2. Depending on the consumption, one can make small aliquots of the solution.

STORAGE
Solution can be stored at 15 – 25 °C (room temperature) for several months.

Precaution:
Discard solution if there is a considerable amount of precipitates.

APPLICATIONS

• Agarose gel electrophoresis of DNA
• Non-denaturing agarose gel electrophoresis of RNA (MOPS buffer is used for denaturing gels)
• Polyacrylamide gel electrophoresis of nucleic acid (both non-denaturing or denaturing)
• DNA automated sequencing gel
• Electrophoretic Mobility Shift Assay (EMSA)

Follow the table to prepare a 5X TBE electrophoresis buffer of various volumes.
Reagents / Volume	100 ml	250 ml	500 ml	1000 ml
Tris base	5.4 g	13.5 g	27 g	54 g
Boric acid	2.75 g	6.9 g	13.75 g	27.5 g
0.5 M EDTA (pH 8.0)	2 ml	5 ml	10 ml	20 ml
Water	Adjust to the final volume 100 ml	Adjust to the final volume 250 ml	Adjust to the final volume 500 ml	Adjust to the final volume 1000 ml

REFERENCES

• Cold Spring Harbor Protocols: Recipe TBE buffer; Full-Text Link: cshlp-pdb.rec8458. URL: http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8458.full?text_only=true 

CALCULATOR

Use this calculator to calculate the quantities of different ingredients to prepare the desired volume of 5x TBE electrophoresis buffer.

mL (Change the volume) Click

To prepare 1000 ml of 5x TBE buffer, you need to dissolve 54 g Tris base (C4H11NO3, Molecular Weight: 121.14), 27.5 g Boric acid (H3BO3, Molecular Weight: 61.83) and 20 ml of 0.5 M EDTA stock solution (pH 8.0) in water to a final volume of 1000 ml.