Preparation of 1 M Tris.Cl buffer, pH 7.2

Preparation of 1 M Tris.Cl buffer, pH 7.2

Overview

  • Tris is also known as THAM, Tris base, Trizma, Tris(hydroxymethyl)aminomethane
  • Tris is one of the most frequently used buffers in cell and molecular biology experiments. It has a pKa value of 8.06 at 25°C and effective pH range between 7.0 and 9.0, which perfectly corresponds to physiological pH range.
  • It is comparatively cheap and highly soluble in water and is easily available in highly purified form. In addition, concentrated solution of Tris can be stored at room temperature for a long time.
  • The pH of Tris solution is dependent of temperature (pH value decreases by ≈0.03 unit per °C increase in temperature) and solution concentration (pH value decreases by ≈0.1 unit when diluted from 100 mM to 10 mM ).
  • Tris is a primary amine, therefore, can not be used in combination with chemicals like formaldehyde and glutaraldehyde for cell and tissue fixation. Tris has also been shown to react with glyoxal.
  • Tris is toxic to many mammalian cell types.

Requirement

  • Reagents
    • Tris base (C4H11NO3, CAS Number: 77-86-1)
    • Conc HCl
    • Deionized / Milli-Q water
  • Equipment and disposable
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer
    • pH meter

Composition: 1 M Tris.Cl

Objective:

Preparation of 1000 ml of 1 M Tris.Cl, pH 7.2

Prior to start: Calibrate the pH meter with pH standard buffers

Preparation

Step 1: To prepare, 1000 ml of 1 M Tris.Cl buffer, weigh out 121.14 gram Tris base (molecular weight = 121.14) and transfer to a 1 litre beaker/conical flask. Dissolve Tris base in 800 ml deionized/Milli-Q water using magnetic stirrer.

Tips:
  • One can use manual shaking using a glass stirring rod. Magnetic stirrer makes the dissolving process easy and convenient.
  • If you want, you can heat the solution (60°C) to accelerate the dissolving process.
Precautions:
  • Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in a solvent.
Step 2: Adjust pH to 7.2 with concentrated HCl.

The solution will have pH between 10.0 – 11.5. Add concentrated HCl (≈ 45 ml) to adjust pH to 7.2.

Tip:
  • You can set desired pH (between 7 – 9) of the solution at this point with the addition of HCl.
Precautions:
  • Don’t adjust pH when the solution is hot. Cool down the solution to room temperature.
  • Take appropriate safety measures (use lab coat, wear gloves) to protect yourself while handling concentrated HCl.
  • Be sure that pH meter is functioning properly. Calibrate pH meter with pH standard buffers in case of any doubt.
  • Silver-containing single-junction pH electrodes (e.g., silver chloride electrode) are incompatible with Tris (Ag-tris precipitation clogs the junction).
Step 3: Adjust the volume to 1000 ml with deionized/Milli-Q water.

To adjust the volume 1000 ml, transfer solution to a 1000 ml measuring cylinder and add deionized/Milli-Q water to bring final volume to 1 liter.

Step 4: Autoclave the solution for 20 minutes at 15 lb/sq.in. on liquid cycle.
Note:
  • A 1 molar solution of Tris buffer (pH 7.2) is clear and colourless.
  • Discard If the solution appear yellowish. Yellowish colour is mostly due to impurities. Use high grade Tris base, suitable for molecular biology work, for solution preparation.

Storage:

The solution can be stored at room temperature for several weeks.

Applications:

The solution is used as a buffer for several biological applications. Some of the most common applications are

  • Preparation of TAE
  • Preparation of TBE
  • Preparation of TE
Follow the table to prepare Tris-Cl, pH 7.2 of specific volume (100 ml, 250 ml, 500 ml and 1,000 ml) and molarity (100 mM, 250 mM, 500 mM and 1M).
Conc. / Volume 100 ml 250 ml 500 ml 1,000 ml
10 mM 0.12 g 0.3 g 0.606 g 1.21 g
100 mM 1.21 g 3.03 g 6.06 g 12.1 g
500 mM 6.057 g 15.14 g 30.285 g 60.57 g
1 M 12.114 g 30.285 g 60.57 g

121.14 g