Preparation of 10X TB (Tris-Borate) electrophoresis buffer

OVERVIEW

EDTA in electrophoresis buffer, although protects DNA from metal-dependent nuclease by sequestering divalent metal ions, also causes heat generation during electrophoresis. To reduce the heat generation during electrophoresis, EDTA can be eliminated from the TBE buffer. The electrophoresis buffer without EDTA, the TB electrophoresis buffer, can be used to perform electrophoresis at high voltage that helps to run the sample quickly (≈ 3 times faster) with better resolution of DNA, especially low molecular weight DNA (Sanderson et al., 2014).

A 10x Tris-Borate (TB) electrophoresis buffer can be prepared by dissolving 108 g Tris base, and 55 g Boric acid in water to a final volume of 1000 ml. 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)
Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

COMPOSITION

Composition of 10X TBE electrophoresis buffer
0.89 M Tris borate
pH 8.2 – 8.4 (at 25°C)

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

OBJECTIVE

Preparation of 1000 ml of 10X TBE electrophoresis buffer, pH 8.3.

PROCEDURE

Step 1: To prepare 1000 ml of 10x TBE buffer, weigh out 108 g Tris base, and 55 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.

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 10X TBE electrophoresis buffer of various volumes.
Reagents / Volume100 ml250 ml500 ml1000 ml
Tris base10.8 g27 g54 g108 g
Boric acid5.5 g13.75 g27.5 g55 g
WaterAdjust to the final volume 100 mlAdjust to the final volume 250 mlAdjust to the final volume 500 mlAdjust to the final volume 1000 ml

REFERENCES

  • Sanderson et al., 2014. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis. Anal Biochem. 454, 44-52. PMID-24637158; Full-Text Links: Sciencedirect, PMC4021863  (download PDF)

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