Preparation of 5X TBE electrophoresis buffer

Overview

  • TBE is one of the very common electrophoresis buffers, used for agarose gel analysis of DNA.
  • It contains Tris, Boric acid and EDTA.
  • Tris-borate provides electrical conductivity as well as maintains the pH.
  • EDTA inhibits metal dependent nucleases by chelating the divalent cations (Ca2+, Mg2+), thus protecting the DNA from nucleases during the run.
  • TBE buffer has better buffering capacity then TAE, therefore it can be used for extended and repeated electrophoresis.
  • Borate from TBE buffer is reported to interact with DNA, which may cause problem in subsequent enzymatic modification of DNA fragment eluted from gel. In such cases, use of TBE buffer should be avoided. TAE is suitable buffer for this purposes.

Requirement

  • Reagents
    • 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 disposable
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

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.

Preparation

Step 1: To prepare 1000 ml of 5X TBE buffer, weigh out 54 gram Tris base and 27.5 gram boric acid. Transfer them to 2 L beaker / conical flask. Add 800 ml deionized / Milli-Q water. Mix until all the ingredients dissolve completely.

Tips:

  • One can use manual shaking using a glass pipette to mix the ingredients. 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.

Precautions:

  • Since pH is dependent on temperature, we recommend to adjust 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.

Optional : One can filter the solution to remove any undissolved materials.

Step 4: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).

Note(s):

  • One can sterilize the solution by passing through 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. Moreover, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

Tips:

  • Transfer the solution to autoclavable bottle.
  • Depending on the consumption, one can make small aliquots of solution.

Storage

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

Precautions:

  • 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)

 

To prepare 5X TBE electrophoresis buffer of various volume (100 ml, 250 ml, 500 ml and 1000 ml), follow the table.
Reagents / Volume 100 ml 250 ml 500 ml 1000 ml
Tris base 5.4 gm 13.5 gm 27 gm 54 gm
Boric acid 2.75 gm 6.9 gm 13.75 gm 27.5 gm
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