Youssef Farhat, Written 5/2/13, Last updated 3/26/14
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Introduction
The purpose of this protocol is to prepare a 1.0 M Tris-HCl stock solution at pH 7.4. Tris-HCl is a buffer that can be used to control the pH of many solutions, including buffers used in ELISAs, cell and tissue lysis buffers, and buffers for fluorogenic assays.
Tris-HCl can be prepared using Tris base (molecular weight: 121.14 g/mol), or Tris-HCl (Tris base which is already combined with HCl in a 1:1 molar ratio, so the molecular weight is 157.6 g/mol). It is important to note which form you use to make the solution, because the initial pH of the solution you make depends on which form you use.
In this protocol, we will use Tris base (MW 121.14 g/mol) to make 500 mL of 1.0 M Tris-HCl, pH 7.4. If you would like to use a similar approach to make a different volume of 1 M Tris-HCl, pH 7.4, then you might find our Reagents Calculator useful.
Materials
- Tris Base (J.T. Baker, #X171)
- 12 M HCl Solution (Sigma-Aldrich, #H1758 – Note: Wear gloves, eye protection and exercise extreme caution when handling this strong acid solution. Aside from being dangerous, it also has a strong smell, so working under a fume hood is ideal.)
- Deionized water
- Normal lab equipment, such as pipettes, serological pipettes, graduated cylinders, beakers, a stir plate, a stir bar, a pH meter, a scale
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Procedure
- Add 60.57 g of Tris Base to an appropriate beaker (500 mL beaker in this case).
- Add 350 mL of deionized water to the beaker.
- Add a stir bar to the beaker and leave it on a stir plate until completely dissolved (~1 min).
- Begin monitoring pH of the solution. It should be basic (pH >7.4).
- Slowly add 32.5 mL of 12 M HCl, while monitoring the pH (Caution: wear gloves, eye protection and exercise extreme caution with this acid solution)
- Add more HCl as necessary until the pH of the solution reaches 7.4
- Once the pH of the solution is 7.4, add enough deionized water to raise the volume to 500 mL.
- Sterile-filter, if possible, and store in the refrigerator for up to 4 months.
Citing this Protocol
Farhat, Y. “Reagent Preparation: Tris-HCl (1.0 M, pH 7.4.” Protocol Place. May 2013. Accessed on mm/dd/yyyy. <http://protocol-place.com>
