TBE buffer (Tris-Borate-EDTA buffer) 1x, 5x & 10x

Introduction

The TBE buffer / Tris Borate EDTA buffer was first reported in 1968, utilizing RNA electrophoresis^[1]. It was later used in other applications containing RNA sequencing^[2] or Maxam-Gilbert’s method of DNA sequencing^[3].

TBE buffer is competing in electrophoretic use with TAE (Tris-Acetate-EDTA). TBE is useful in separating short fragments <1.5kbp (Biogen) and has a significantly higher buffering capacity, but the borate involved in this buffer could potentially inhibit the activity of enzymes. Thus, TAE is a better choice if the electrophoresis is followed by nucleic acid isolation from the gel and other steps (e.g., ligation)^[4]. The electrophoresis with TAE is also thought to be faster than TBE for long fragments but slower for short fragments <300bp^[5].

Other less-used alternatives have been proposed. LB (lithium borate) is a buffer with lower conductivity. Therefore, it can be used with higher voltage and fast up the electrophoresis. However, the price of lithium salts is high, so it was replaced by sodium (SB), resulting in similar physical properties and a lower price^[6].

Applications

Commonly used for short fragment electrophoresis of DNA/RNA as 1X and 0.5X solution.

Composition

The TBE is commonly prepared as 5X and 10X stock solutions. The 5X is preferred by some labs because it precipitates less than 10X.

Reagent	Molecular weight	1X molarity ²	5X molarity	10X molarity	Add for 1 of 5X	Add for 1L of 10X
Tris base¹	121.14	90 mM	450 mM	900 mM	54.51 g	109.03 g
Boric acid	61.83	90 mM	450 mM	900 mM	27.82 g	55.65 g
Na₂EDTA ³	336.21 (anhydrous)	2 mM	10 mM	20 mM	3.36 g	6.74 g
	372.24 (dihydrate)				3.72 g	7.45 g

Table 1. Composition of TBE buffer.

¹Tris base is a trivial name for tris(hydroxymethyl)aminomethane.

²Sometimes, the 0.5X working concentration is used. It has lower conductivity but a lower buffering capacity.

³Use either anhydrous or dihydrate. If you have EDTA solution, you can use it. It would spare you time when you would wait for EDTA to dissolve. For example, use 20 ml of 0.5 M EDTA (pH 8.0) for 1l of 5X, or 40ml for 1l of 10X.

Learn how to make EDTA solution here.

pH

The effective range of Tris is 7-9. The effective range of boric acid is 8-10. Concluding this information, the overlap of buffering properties of both is 8-9, thus mostly used pH 8.3 was found to be optimal.

NaOH/HCl is used for adjustment.

The preparation of buffer without adjustment is reproducible. Keep in mind that pH meters (uncalibrated) work with some error and therefore, it is sometimes better to use non-adjusted buffers if the pH of dissolved compounds is nearby the expected value.

Preparation

Put ¾ intended volume of distilled water into beaker/flask.
Prepare the exact amounts of compounds using a weighing balance. If you have EDTA solution, you can measure the volume and mix it directly with water.
Dissolve the reagents in water. The pH should raise while dissolving the Tris base. The higher pH will help EDTA to dissolve (if you used crystalline EDTA) but it is still a slow process. The magnetic stirrer is really helpful in this step.
Adjust the pH (if needed).
Add distilled water to final volume.
Autoclave or filter the solution (Especially, if you intend to use it with RNA).

Storage

Up to 3 months at room temperature (in the fridge may result in precipitation).

Points to be noted

Boric acid is toxic. Therefore, use gloves and omit contact with it.
Tris base is a skin and eye irritant. EDTA is an eye irritant.

Reference

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