Preparation of 1 M Tris.Cl Buffer

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 an effective pH range between 7.0 and 9.0, which perfectly corresponds to the physiological pH range.
  • It is comparatively cheap and highly soluble in water and is easily available in highly purified form. In addition, concentrated solutions of Tris can be stored at room temperature for a long time.
  • The pH of the Tris solution is dependent on 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 disposables
> Measuring cylinder
> Conical flask / Beaker
> Magnetic stirrer
> pH meter

Composition: 
1 M Tris.Cl
pH (can set any desirable pH between 6.8 to 8.5)

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

Preparation

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

Tips:
One can use manual shaking using a glass stirring rod. The magnetic stirrer makes the dissolving process easy and convenient.
If you want, you can heat the solution (60°C) to accelerate the dissolving process.

Precaution:
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 a pH between 10.0 – 11.5. Add concentrated HCl (≈ 45 ml) to adjust pH to 7.2.

Tip:
You can set the desired pH (between 6.8 – 9) of the solution at this point with the addition of HCl.

Precautions:
1. Don’t adjust the pH when the solution is hot. Cooldown the solution to room temperature.
2. Take appropriate safety measures (use a lab coat, wear gloves) to protect yourself while handling concentrated HCl.
3. Be sure that the pH meter is functioning properly. Calibrate pH meter with pH standard buffers in case of any doubt.
4. 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 the solution to a 1000 ml measuring cylinder and add deionized/Milli-Q water to bring the final volume to 1 liter.

Step 4: Autoclave the solution for 20 minutes at 15 lb/sq.in. on liquid cycle.

Notes:
1. A 1 molar solution of Tris buffer (pH 7.2) is clear and colorless.
2. Discard If the solution appears yellowish. The yellowish color is mostly due to impurities. Use a 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, and molarity.
Conc. / Volume100 ml250 ml500 ml1,000 ml
10 mM0.12 g0.3 g0.606 g1.21 g
100 mM1.21 g3.03 g6.06 g12.1 g
500 mM6.057 g15.14 g30.285 g60.57 g
1 M12.114 g30.285 g60.57 g121.14 g

Molarity calculator: Calculate the amount of Tris Base (M.W. 121.14 g/mol) required to prepare a solution of specific molarity and volume

Molecular weight of Tris Base: 121.14 g/mol


Molarity of Tris solution: M

(Change the molarity of the solution)

 


Volume of stock solution: ml

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Required amount of Tris: 121.14g

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