20X DPBS Stock Solution

Youssef Farhat, Written 5/2/13, Last updated 8/1/13

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Introduction

The purpose of this protocol is to prepare a 20X DPBS stock solution which can then be diluted down to 1X when needed.  DPBS, or Dulbecco’s Phosphate-Buffered Saline, is often used as a neutral, isotonic buffer for cell culture and in assays such as ELISAs.  Be sure to sterilize DPBS if used for cell culture, or purchase a bottle of sterile DPBS from a company (such as: Gibco, #14190).

In this protocol, we will use solid NaCl (molecular weight: 58.44 g/mol), KCl (MW: 74.55 g/mol), KH₂PO₄ (MW: 136 g/mol), and Na₂HPO₄ (MW: 142 g/mol) to make 1 L of 20X DPBS stock solution.

Materials

• NaCl (VWR, #BDH0286)
• KCl (MW: 74.55 g/mol),
• KH₂PO₄ (MW: 136.09 g/mol)
• Na₂HPO₄ (MW: 141.96 g/mol)
• 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.)
• NaOH pellets (J.T. Baker, #3722)
• Deionized water
• Normal lab equipment, such as pipettes, serological pipettes, graduated cylinders, beakers, a stir plate, a stir bar, a scale, pH meter

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Procedure

1. Add the following to a 1 L beaker:
   1. 160 g of NaCl
   2. 4 g of KCl
   3. 4 g of KH₂PO₄
   4. 23 g of Na₂HPO₄
2. Add 800 mL of deionized water to the beaker.
3. Add a stir bar to the beaker and leave it on a stir plate until it completely dissolves (10 min)
4. Check the pH with a pH meter to ensure that the solution’s pH is 7.4.
   1. Adjust the pH, if necessary, with HCl (exercise extreme caution!) or NaOH pellets.
5. Raise the volume to 1 L with deionized water.
6. Sterile-filter, if desired, and transfer to an appropriately labeled container.

Citing this Protocol

Farhat, Y. “Reagent Preparation: 20X DPBS.” Protocol Place. May 2013. Accessed on mm/dd/yyyy. <http://protocol-place.com>