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

Table of Contents
    Add a header to begin generating the table of contents

    Introduction

    The TBE buffer / Tris Borate EDTA buffer is 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 the separation of short fragments <1.5kbp (Biogen) and has a significantly higher buffering capacity, but 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].

    Uses

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

    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.

    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.

    ReagentMolecular weight1X molarity 25X molarity10X molarityAdd for 1 of 5XAdd for 1L of 10X
    Tris base1121.1490 mM450 mM900 mM54.51 g109.03 g
    Boric acid61.8390 mM450 mM900 mM27.82 g55.65 g
    Na2EDTA 3336.21 (anhydrous)2 mM10 mM20 mM3.36 g6.74 g
    372.24 (dihydrate)3.72 g7.45 g

    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.

    Reagents

    • Tris (tris(hydroxymethyl)aminomethane)
    • Boric acid
    • Na2EDTA
    • HCl (if adjustment needed)
    • Distilled water

    Instruments and other requirements

    • Glass beaker
    • Weighing balance
    • Magnetic stirrer and pellet
    • pH meter
    • Measuring cylinders
    • Pipette

    Procedure

    1. Put ¾ intended volume of distilled water into beaker/flask.
    2. 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.
    3. 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.
    4. Adjust the pH (if needed).
    5. Add distilled water to final volume.
    6. 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).

    Caution

    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.

    References

    1. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels.
    2. Studies on polynucleotides cxxii. The dodecanucleotide sequence adjoining the c-c-a end of the tyrosine transfer ribonucleic acid gene.
    3. Sequences of Controlling Regions of the Lactose Operon. Cold Spring Harbor Symposia on Quantitative Biology
    4. ThoughtCo
    5. TBE, or not TBE; that is the question: Beneficial usage of tris-borate for obtaining a higher resolution of small DNA fragments by agarose gel electrophoresis.
    6. Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.
    7. Biogen.

    Leave a Comment