Tris-HCl Solution (pH 7.5) Protocol

Tris-HCl Solution (pH 7.5) Protocol

Created By: Jason Gardiner

1. Purpose:

This protocol details the preparation of a 1M Tris-HCl solution buffered to pH 7.5, commonly used in molecular biology techniques.

2. Materials:

• Chemicals:
  • Tris base (molecular weight: 121.14 g/mol)
  • Concentrated Hydrochloric acid (HCl, approximately 37%)
• Equipment:
  • Analytical balance
  • Magnetic stirrer and stirring bar
  • Graduated cylinder (500 ml)
  • Volumetric flask (500 ml)
  • pH meter with calibration solutions
  • Beaker
  • Pipette
• Personal Protective Equipment (PPE):
  • Lab coat
  • Gloves
  • Safety glasses

3. Procedure:

1. Preparation: Clean and dry all glassware thoroughly.

2. Dissolving Tris base:
   • Weigh 60.55 g of Tris base using the analytical balance.
   • Transfer the weighed Tris base to a clean beaker.
   • Add approximately 400 ml of distilled water to the beaker.
   • Place the beaker on a magnetic stirrer and stir continuously until the Tris base is fully dissolved.
3. pH Adjustment:
   • Carefully calibrate the pH meter using standard calibration solutions according to the manufacturer’s instructions.
   • Slowly add concentrated HCl to the Tris solution while stirring continuously, monitoring the pH with the calibrated pH meter.
   • Aim to adjust the pH to 7.5. The amount of HCl required (approximately 65 ml) is an estimate and may vary slightly depending on factors like the purity of the chemicals.

4. Final Volume Adjustment:

   • Carefully transfer the Tris-HCl solution to a clean 500 ml volumetric flask.
   • Rinse the beaker with distilled water and add the rinsings to the volumetric flask to ensure complete transfer.
   • Add distilled water to the volumetric flask until the meniscus reaches the 500 ml mark. Mix thoroughly by inverting the flask several times.
5. Sterilization: Autoclave the Tris-HCl solution at 121°C for 15 minutes to sterilize it.

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4. Storage: Store the sterilized Tris-HCl solution in a tightly sealed container at room temperature. When stored properly, it is typically stable for several months.