Tris-borate-EDTA (TBE) buffer is often used for agarose gel electrophoresis in the analysis of DNA products resulting from PCR amplification, DNA purification protocols, or DNA cloning experiments. It is particularly useful for separation of smaller DNA fragments (MW < 1000), for example small products of restriction enzyme digests. TBE has a greater buffering capacity and will give sharper resolution than TAE buffer. TBE is generally more expensive than TAE, and inhibits DNA ligase which may cause problems if subsequent DNA purification and ligation steps are intended.
Time Required: 30 minutes
- Prepare a Stock Solution of EDTA
An EDTA (ethylenediamine tetraacetic acid) solution is prepared ahead of time. EDTA will not go completely into solution until the pH is adjusted to about 8.0. For a 500 mL stock solution of 0.5 M EDTA, weigh out 93.05 g EDTA disodium salt (FW = 372.2). Dissolve in 400 mL deionized water and adjust the pH with NaOH. Top up the solution to a final volume of 500 mL.
- Prepare a Stock Solution of TBE
Make a concentrated (5x) stock solution of TBE by weighing 54 g Tris base (FW = 121.14) and 27.5 g boric acid (FW = 61.83) and dissolving both in approximately 900 mL deionized water. Add 20 mL of 0.5 M EDTA (pH 8.0) and adjust the solution to a final volume of 1 L. This solution can be stored at room temperature but a precipitate will form in older solutions. Store the buffer in glass bottles and discard if a precipitate has formed.
- Prepare a Working Solution of TBE
For agarose gel electrophoresis, TBE can be used at a concentraion of 0.5x (1:10 dilution of the concentrated stock). Dilute the stock solution by 10x in deionized water. Final solute concentrations are 45 mM Tris-borate and 1 mM EDTA. The buffer is now ready for use in running an agarose gel.
What You Need
- EDTA disodium salt
- Tris base
- Boric acid
- pH meter and calibration standards as appropriate
- 600 mL and 1500 mL beakers or flasks
- Graduated cylinders
- Deionized water
- Stir bars and stir plates