Phosphate Buffered Saline (PBS) composition and preparation

Phosphate buffered saline (a.k.a PBS or PBS buffer) is one of the most used buffers since it is isotonic to most of the cells. We have placed reagents required for preparation in a table. This table also contains information on molarity of each reagent and amount of salt (s) to be added for making 500mL and 1L of 1X and 10X. For your convenience, we have given the molecular weights of each reagent and for it’s most used hydrated forms too.  It is up to the user to determine whether hydrated forms make any difference for the experiments or not.  

There are many different formulations for phosphate buffered saline. Some of them contain calcium and magnesium salts. In this page, we are restricting ourselves to provide the most used PBS composition based on (CSHL protocols and Molecular cloning by Sambrook).

Applications

  • Washing buffer for cells/tissue.
  • Diluent.

pH

Adjust pH to 7.2 or 7.4 (depending on requirement) using 1N HCl and 1N NaOH

Composition

Phosphate buffered saline (PBS) composition and preparation
pH of the buffer to be adjusted to 7.4 with HCl and NaOH.
Phosphate buffered saline (PBS) composition and preparation
pH of the buffer to be adjusted to 7.4 with HCl and NaOH.
Reagent M.W Molarity of 1X Add for 500 ml 10X Add for 1 L of 10X Add for 500 ml 1X Add for 1 L of 1X
NaCl 58.44 137 mM 40 g 80 g 4 g 8 g
KCl ‎74.54 2.7 mM 1 g 2 g 0.1 g 0.2 g
Na2HPO4 141.96 10 mM 7.2 g 14.4 g 0.72 g 1.44 g
Na2HPO4 2H2O 177.99 10 mM 8.9 g 17.8 g 0.89 g 1.78 g
Na2HPO4 7H2O 268.07 10 mM 13.4 g 26.8 g 1.34 g 2.68 g
Na2HPO4 12H2O 358.14 10 mM 17.9 g 35.8 g 1.79 g 3.58 g
KH2PO4 136.08 1.8 mM 1.2 g 2.4 g 0.12 g 0.24 g

Instruments and other requirements

  • Weighing balance.
  • Graduated cylinder and a glass beaker.
  • Magnetic stirrer and magnetic bead/pellet.
  • pH meter.
  • 1N HCl and 1N NaOH for pH adjustment.

Procedure

Use ddH2O only for making PBS.

  1. Determine how much volume (500mL or 1L or any other) and concentration (1X or 10X) you want to make.
  2. Weigh the reagents accordingly (Refer to the table above).
  3. Add salts one after the other to the ddH2O (take 400ml or 800ml of initial volume if you want to make 500mL or 1L respectively).
  4. Dissolve salts by using magnetic stirrer.
  5. Adjust pH to 7.4 using HCl and NaOH.
  6. Bring the volume to 500mL or 1L as planned.
  7. Aliquot to smaller volumes if needed (frequent opening and closing in open air might result in microbial growth).
  8. Autoclave for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle or Filter sterilize.

Storage

       You can store PBS (Phosphate buffered saline) at room temperature. Storage of 10X at 4˚C might lead to the formation of crystals.

Reference

  1. Cold Spring Harbor Protocols.

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