Preparing 10x TBE Electrophoresis buffer

Abstract

TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water.

EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary cofactors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg2+ is also a cofactor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low (typically at around 1 mM).

TBE buffer is used to prepare agarose gels and as running buffer in agarose gel electrophoresis to separate and identify and analyse nucleic acids.

SCOPE:

This protocol covers the steps involved in making 1L of 10x TBE buffer from Tris, Borate and EDTA powders

DEFINITIONS:

TBE: Tris Borate EDTA

Steps

Preparing 10x TBE buffer solution (1 liter)

Use an electronic balance to weigh out 108g Tris base (CAS# 77-86-1, free base) into a 1000mL ml or higher volume beaker ( depending on what volume of beaker is available and should be big enough to contain the powders to be dissolved).

Weigh 55g boric acid (CAS# 10043-35-3) into the same beaker

Weigh9.3g EDTA, disodium salt dihydrate (Cas# 6381-92-6, C₁₀H₁₄N₂Na₂O₈· 2H₂O, MW: 372.24) into a weighing boat and pour into the beaker.

Note

Note that you can also use 0.5Molarity (M) EDTA solution at 8.3. In this case skip to Step 4. Then add 40mL EDTA solution to the dissolved salts.

Measure 100mL distilled or deionised water using a measuring cylinder, pour into the 1000mL beaker and stir for 0h 1m 0s either manually or using a magnetic stirrer and flea.

Note

If the TBE buffer will be used for RNA work, ensure that RNase-free water is used.

Adjust volume to 1L by adding 900mL of distilled or deionised water.

Place the beaker on top of a magnetic stirrer and put a magnetic flea in the solution. Allow to stir until you have a clear solution, this may take up to 0h 30m 0s

Check the pH using a pH meter or Universal indicator strips and record the result for quality control. A properly prepared solution should be 8.3 and should not require adjustment.

Autoclave (if sterile solution is needed) and pour solution into clean, autoclaved and labelled glass bottles.

Note

10 x TBE buffer is stable for up to 6 months at room temperature. The solution should be discarded if it becomes cloudy.

Make 1 x TBE working solution

This buffer can be diluted to 1x each time before use by diluting 100mL of 10x TBE with 900mL of distilled water to have 1L of 1x TBE buffer.

Abstract

Steps

Preparing 10x TBE buffer solution (1 liter)

Make 1 x TBE working solution

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