Preparation of 1 M Tris.Cl buffer, pH 7.2
- 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.
- 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
Preparation of 1000 ml of 1 M Tris.Cl, pH 7.2
Prior to start: Calibrate the pH meter with pH standard buffers
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.
- 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.
- 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.
The solution will have pH between 10.0 – 11.5. Add concentrated HCl (≈ 45 ml) to adjust pH to 7.2.
- You can set desired pH (between 7 – 9) of the solution at this point with the addition of HCl.
- 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).
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.
- 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.
The solution can be stored at room temperature for several weeks.
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||