Preparation of 50X TAE Electrophoresis Buffer

OVERVIEW

  • TAE (Tris-Acetate EDTA) electrophoresis buffer 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 solution 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 a lower buffering capacity than TBE, therefore the use of TAE should be avoided for extended and repeated electrophoresis.
  • A 50x TAE buffer can be prepared by mixing and dissolving 242 g Tris base, 100 ml of 0.5 M EDTA and 57.1 ml glacial acetic acid in a deionized water to a final volume of 1000 ml. The pH of the final solution should be between 8.2 – 8.4. 

Preparation of 50X TAE electrophoresis buffer

REQUIREMENTS

Reagents and solutions
Tris base (C4H11NO3, Molecular Weight: 121.14)
Glacial acetic acid * (CH3COOH, Molecular Weight: 60.05, Molarity: 17.5M)
0.5 M EDTA stock solution (pH 8.0)
Deionized / Milli-Q water

* The molarity of glacial acetic acid (100% acetic acid) is ≈ 17.5 M. (see how to calculate Molarity of Glacial Acetic Acid)

Equipment and disposables
Measuring cylinder
Conical flask / Beaker
Magnetic stirrer

COMPOSITION

Composition of 50x TAE buffer (Stock Solution)
2.0 M Tris base
1.0 M Acetic acid

0.05 M EDTA
pH 8.2 – 8.4 (at 25°C)

Composition of 1x TAE buffer (Working Solution)
40 mM Tris base
20 mM Acetic acid
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 g 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.

Tip
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.

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.

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), 121-124°C on liquid cycle).

Tips
1. Transfer the solution to an autoclavable bottle before autoclaving.
2. 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.

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

Preparation of 1x TAE electrophoresis buffer from 50x concentrated stock solution:

Take 1 volume of concentrated stock solution and add 49 volumes of distilled water. Mix. For example, to prepare 500 ml of 1x TAE solution from a 50x stock solution, take 490 ml water in a measuring cylinder. Add 10 ml of 50x concentrated stock solution and mix.

APPLICATIONS

Agarose gel electrophoresis of DNA
TAE buffer is suitable for applications where gel eluted DNA fragments need to be modified using DNA modifying enzymes. In such cases, the use of TBE buffer should be avoided as the borate of TBE buffer inhibits many enzymes (e.g., DNA ligases).

Follow the table to prepare a 50x TAE electrophoresis buffer of various volumes.
Reagents / Volume100 ml250 ml500 ml1000 ml
Tris base24.2 g60.5 g121 g242 g
Glacial acetic Acid5.71 ml14.27 ml28.55 ml57.1 ml
0.5 M EDTA (pH 8.0)10 ml25 ml50 ml100 ml
WaterAdjust the final volume to 100 mlAdjust the final volume to 250 mlAdjust the final volume to 500 mlAdjust the final volume to 1000 ml

CALCULATOR: 

Use calculator to calculate the required amount of ingredients to prepare specific volume of 50x TAE electrophoresis buffer

ml

To prepare 1000 ml of 50x TAE electrophoresis, you need
242.28 g Tris
57.1428 ml Glacial acetic acid
100 ml of 0.5M EDTA solution

REFERENCES

Cold Spring Harbor Protocols: Recipe TAE; Full-Text Link: cshprotocols-pdb.rec8644. URL: http://cshprotocols.cshlp.org/content/2006/1/pdb.rec8644.full

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