TAE (Tris-acetate-EDTA) buffer, is named so because it contains the three ingredients Tris base, acetic acid, and EDTA; it is a solution that is widely used as an electrophoresis running buffer and for the formation of agarose gels. Tris-acetate inhibits DNA hydrolysis, whereas EDTA, a cation chelator, protects nucleic acids from enzymatic degradation.
The ionic strength and buffering capacity of this buffer are both modest. It is best suited to electrophoresis of big (>20 kilobases) chunks of DNA and must be changed or recirculated on a regular basis for longer (>4 hours) gel run periods. Considering this, one might want to make multiple batches of the buffer. Given how simple the buffer is to construct and how fast the procedures can be completed, producing more than one batch at a time should not be very time-consuming or complex.
Required Reagents For the Preparation of TAE Buffer
- EDTA Solution (ethylene diamine tetra-acetic acid),
- Disodium salt,
- Tris base, and
- Glacial acetic acid.
Equipment Required For the Preparation of TAE Buffer
- pH meter and calibration standards
- 600ml to 1500ml capacity beakers or flasks
- Graduated cylinders
- Deionized water
- Stir bars
- Stir plates
Preparing Stock Solution of EDTA
An EDTA Solution is made beforehand. Until the pH is brought down to roughly 8.0, EDTA will not dissolve entirely in a solution.
- Weigh 93.05 grams of EDTA disodium salt (FW = 372.2) to make a stock solution with 0.5 M (molarity, or concentration), which will be 500 ml in volume.
- It should be dissolved in 400 milliliters of deionized water before using sodium hydroxide (NaOH) to correct the pH.
- Add more solution until the total volume is 500 ml.
Preparing 50x TAE Buffer Stock
|Chemical||Weight / Volume||Molarity|
|0.5M EDTA pH 8.0||100 mL||50 mM millimolar|
|Acetic Acid||57.1 mL|
|Tris Base||242 gm||2 Molar|
|Milli-Q||Up to 1000 mL|
- To make a concentrated (50x) TAE stock solution, weigh out 242 grams of Tris base (FW = 121.14) and dissolve it in 750 milliliters of deionized water.
- Carefully mix in 57.1 mL of glacial acid and 100 mL of 0.5 M EDTA (pH 8.0).
- The solution should now be adjusted to a final volume of 1 liter.
- This stock solution is suitable for storing at room temperature.
- This buffer’s pH is not altered and should be about 8.5.
Preparing 1x TAE Buffer Solution
The working solution of 1x TAE buffer is simply prepared by diluting the stock solution by 50x in deionized water.
- The final solute concentrations are 40 mM (millimolar) Tris-acetate
- 1 mM EDTA.
The buffer is now ready for use in conducting an agarose gel.
Calculation For Preparing 1x TAE From 50x TAE Stock
The following formula may be used to calculate this:
C1 x V1 = C2 x V2 Where, C1 - Initial Concentration V1 - Initial Volume C2 - Final Concentration V2- Final Volume
For example, how much 50X TAE is required to produce 1X TAE Buffer from 50X TAE Buffer Stock?
C1 x V1 = C2 x V2
or, 50 x V1 = 1 x 300
or, V1 = 300 / 50
V1 = 6mL
To generate 300mL of 1X TAE Buffer, use 6mL of 50X TAE Stock solution and dilute it with Milli-Q water (6mL of 50X TAE + 294mL of Water).
Application of TAE Buffer
- The 1x TAE buffer is used in both the agarose gel and as a running buffer.
- TAE buffer has been employed for agarose gel electrophoresis of RNA.
- A study of free DNA solution mobility in TAE at various buffer concentrations in the presence and absence of added NaCl has been published.