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Preparation of 5X TBE electrophoresis buffer

Overview

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Preparation of 50X TAE electrophoresis buffer.htm

Requirement

  • Equipments and disposables
    • 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 25C)
Composition of 1X TBE electrophoresis buffer
  • 89 mM Tris borate
  • 2 mM EDTA
  • pH 8.2 - 8.4 (at 25C)
Objective
  • Preparation of 1000 ml of 5X TBE electrophoresis buffer, pH 8.3.

Preparation

  • To prepare 1000 ml of 5X TBE buffer, weigh 54 gm Tris base and 27.5 gm boric acid. Transfer them to 2 L beaker / conical flask. Add 800 ml deionized / Milli-Q water. Add 20 ml of 0.5 M EDTA solution. Mix until all the ingredients dissolve completely.
    Tip(s):
    1. One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.
    2. We recommend to add 0.5 M EDTA solution after adding water.
    Precaution(s):
    Do not dissolve in 980 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent. see solution preparation

  • Adjust the pH 8.3 if required.
    Precaution(s):
    Since pH is dependent on temperature, we recommend to adjust the solution pH at room temperature (25C).

  • Adjust the solution volume to 1000 ml with deionized / Milli-Q water. Mix it again.

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

  • Transfer the solution to autoclavable bottle.
    Tip(s):
    Depending on the consumption, one can make small aliquots of solution.

  • Sterilize the solution by autocaving (20 minutes at 15 lb/sq.in. (psi) from 121-124C on liquid cycle).
    Note(s):
    One can sterilize the solution by passing through 0.22 μ filter unit. Filter strilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. Morover, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorgasnisms including mycoplasma.


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

Applications
Preparation of 1000 ml of 5X TBE electrophoresis buffer
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
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