Tris Buffer

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    Introduction:

    Tris (hydroxymethyl) aminomethane is also known as THAM, with pKa 8.06. Tris (M.W: 121.14) is a white crystalline powder having molecular formula C4H11NO3. It forms a clear solution in water. Tris is used as a buffering system in most of the biological experiments.

    Applications:

    Tris is a component of:

    • DNA extraction buffer (Edwards buffer)
    • DNA Resuspension (TE)
    • DNA electrophoresis buffer (TAE)
    • Stacking and ruining gel preparations of SDS-PAGE
    • Towbin buffer (the running buffer for SDS-PAGE)

    pH:

    The pH of the Tris buffer is adjusted based on the requirement. Predominantly, the pH range for Tris buffer is 7.2 to 9.0

    When you dissolve Tris base, the pH is more alkaline. To bring down the pH, you may need higher volumes of concentrated HCl. In case of adjusting pH towards the alkaline range, use 1N NaOH.

    Note:

    Tris is one of the most temperature susceptible buffer systems. When you are setting the desired pH, you need to consider the deviation caused by temperature. For increment in each degree Celsius of temperature, pH drops by 0.028 pH units and vice versa. An example better explains the scenario, so let’s consider one.

    You have made Tris buffer with pH to 7.4 at 25oC. For an experiment, you are using the same buffer at 37 oC. Since you have adjusted buffer pH to be 7.4 at 25 oC, at 37 oC, it would be 7.064

    (12oC X 0.028 pH units = 0.336pH unit). So, if you want to use a buffer at 37oC, you have to prepare the buffer with pH 7.736 at room temperature, and it would be 7.4 at 37oC.

    Composition:

    Reagent0.5M Tris buffer (1L)1M Tris buffer (1L)
    Tris base60.57g121.14g
    H2Omake upto 1000 mlmake upto 1000 ml

    Reagents:

    • TRIS base
    • Distilled water
    • Concentrated  HCl
    • 1N NaOH

    Instruments and other requirements

    • Dropper for adding HCl or NaOH
    • Beaker
    • Measuring jar
    • Magnetic stirrers
    • pH meter
    • Reagent bottles

    Preparation:

    1. For 0.5 M (250ml)
    2. Take 200ml of distilled water in 500ml beaker and place it on a magnetic stirrer.
    3. Now add exactly weighed Tris base to the water and allow it to dissolve completely (clear solution)
    4. Now test the pH with pH meter by dipping an electrode into the solution.
    5. Adjust the pH to the desired value.
    6. Make up the volume to 250 ml.

    Storage:

    Tris buffer can be stored at room temperature or at 4°C. Tris buffer is susceptible to bacterial attack. For better and long term storage, it is advisable to store at 4°C after Filter sterilization (recommended) or autoclave.

    References:

    AppliChem

    Wikipedia

    Cold Spring Harbor Laboratory

    2 Comments

    1. Ross Schur on September 3, 2019 at 9:17 pm

      That is a very good tip particularly to those fresh to the blogosphere. Brief but very accurate information… Many thanks for sharing this one. A must read post!

      • Sharebiology team on September 4, 2019 at 5:00 pm

        Hello Ross,

        We are glad you liked it. It means a lot to us.

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