Phosphate Buffered Saline (PBS)

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    Introduction

    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.

    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

    ReagentM.WMolarity of 1XAdd for 500 ml 10XAdd for 1 L of 10XAdd for 500 ml 1XAdd for 1 L of 1X
    NaCl58.44137 mM40 g80 g4 g8 g
    KCl‎74.542.7 mM1 g2 g0.1 g0.2 g
    Na2HPO4141.9610 mM7.2 g14.4 g0.72 g1.44 g
    KH2PO4136.081.8 mM1.2 g2.4 g0.12 g0.24 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 a 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

    Cold Spring Harbor Protocols.

     

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