Preparation of 50X TAE electrophoresis buffer

Overview

  • TAE is one of the very common electrophoresis buffers, used for agarose gel analysis of DNA.
  • It contains Tris, acetic acid and EDTA.
  • Tris-acetate provides electrical conductivity and maintains pH.
  • EDTA inhibits metal dependent nucleases by chelating the divalent cations (Ca2+, Mg2+), thus protecting the DNA from nucleases during the run.
  • TAE buffer has lower buffering capacity than TBE, therefore it’s use should be avoided for extended and repeated electrophoresis.
  • TAE buffer is suitable for applications where gel eluted DNA fragments need to be modified using DNA modifying enzymes. In such cases, use of TBE buffer should be avoided as the borate of TBE buffer inhibits many enzymes (e.g., DNA ligases).

Requirement

  • Reagents
    • Tris base (C4H11NO3, Molecular Weight: 121.14)
    • Glacial acetic acid (CH3COOH, Molecular Weight: 60.05)
    • 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 50X TAE buffer (Stock Solution)

  • 2.0 M Tris acetate
  • 0.05 M EDTA
  • pH 8.2 – 8.4 (at 25°C)

Composition of 1X TAE buffer (Working Solution)

  • 40 mM Tris acetate
  • 1 mM EDTA
  • pH 8.2 – 8.4 (at 25°C)

Objective

  • Preparation of 1000 ml of 50X TAE electrophoresis buffer.

Preparation

Step 1: Weigh out 242 grams of Tris base and transfer it to 2 L beaker / conical flask. Add 750 ml deionized / Milli-Q water and mix until all Tris base dissolves 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 100 ml of 0.5 M EDTA solution and 57.1 ml glacial acetic acid. Mix the solution again. Adjust pH to 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).

Notes:

  • 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 before autoclaving.
  • 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
To prepare 50X TAE 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 24.2 gm 60.5 gm 121 gm 242 gm
Glacial acetic Acid 5.71 ml 14.27 ml 28.55 ml 57.1 ml
0.5 M EDTA (pH 8.0) 10 ml 25 ml 50 ml 100 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