How to Make TBE Buffer in 3 Easy Steps
This buffer is used for electrophoresis separation of DNA
TBE buffer (Tris-borate-EDTA) is a buffer solution made up of Tris base, boric acid and EDTA (ethylenediaminetetraacetic acid). This buffer is often used for agarose gel electrophoresis in the analysis of DNA products which result from PCR amplification, DNA purification protocols or DNA cloning experiments.
TBE buffer is particularly useful for the separation of smaller DNA fragments (MW < 1000), such as small products of restriction enzyme digests. TBE has a greater buffering capacity and will give sharper resolution than TAE buffer. TAE (Tris-acetate-EDTA) buffer is a solution made up of Tris base, acetic acid, and EDTA.
TBE is generally more expensive than TAE and inhibits DNA ligase, which may cause problems if subsequent DNA purification and ligation steps are intended. With the three simple steps that follow, learn how to make TBE buffer. It shouldn't take more than about 30 minutes to create.
What You Need
To make TBE buffer, you'll need just four substances. The remaining items on this list are equipment. The four substances required are EDTA disodium salt, Tris base, boric acid and deionized water.
As for equipment, you'll need a pH meter and calibration standards, as appropriate. Additionally, you'll want some 600-milliliter and 1500-milliliter beakers or flasks. Rounding out your equipment needs are graduated cylinders, stir bars and stir plates.
Check the inventory at the lab you'll be using to make sure you have everything you need before you get started. Nothing's worse than having to stop in the middle of preparing a solution because you've run out of the proper materials.
If your lab is at school or your worksite, check with the proper personnel to see that they have all the items in the stock. Doing so may save you time and energy in the end.
Stock Solution of EDTA
An EDTA solution should be prepared ahead of time. EDTA will not go completely into the solution until the pH is adjusted to about 8.0. For a 500-milliliter stock solution of 0.5 M EDTA, weigh out 93.05 grams of EDTA disodium salt (FW = 372.2). Then dissolve it in 400 milliliters of deionized water and adjust the pH with NaOH. After that, top up the solution to a final volume of 500 milliliters.
Stock Solution of TBE
Make a concentrated (5x) stock solution of TBE by weighing 54 grams of Tris base (FW = 121.14) and 27.5 grams of boric acid (FW = 61.83) and dissolving both in approximately 900 milliliters of deionized water. Then add 20 milliliters of 0.5 M EDTA (pH 8.0) and adjust the solution to a final volume of 1 liter. 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.
Working Solution of TBE
For agarose gel electrophoresis, a TBE buffer can be used at a concentration 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.