Jun 14, 2022

Public workspacePreparation of PBS Solution V.1

This protocol is a draft, published without a DOI.
  • 1Mboalab, Beneficial Bio
Nadine Mowoh
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Protocol Citation: Stephane Fadanka, Mujar Minette Shalo, Nadine Mowoh 2022. Preparation of PBS Solution. protocols.io https://protocols.io/view/preparation-of-pbs-solution-b85dry26
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 10, 2022
Last Modified: June 14, 2022
Protocol Integer ID: 62341
Keywords: PBS, Phosphate Buffered Saline
Abstract
Phosphate buffered saline ( PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. This SOP gives one of the most common formulations used in preparing PBS (1x PBS).

  • 137 mM NaCl
  • 10 mM Na2HPO4
  • 1.8 mM KH2PO4 
  • pH 7.2 -7.4

PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface. It is applied in washing cells after protein expression procedures due to its osmolarity and pH to avoid cell disruption to preserve and subsequently freeze the cells for downstream molecular biology applications. 

Materials
Reagents
  • ReagentSodium chlorideSigma – Aldrich
  • ReagentSodium phosphate dibasic heptahydrateAcros Organics
  • ReagentPotassium phosphate monobasicAcros Organics
  • ReagentSodium hydroxide
  • ReagentHydrochloric acidFisher Scientific
Materials and equipment
  • 500ml Beaker
  • 100ml Measuring Cylinder
  • Distilled water
  • pH Meter
  • Electronic Balance
  • Weighing boat
  • Magnetic Stirrer
  • Refrigerator
  • Autoclave
  • 500ml Duran Bottle





Protocol materials
ReagentSodium hydroxide
ReagentHydrochloric acidFisher Scientific
ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)
ReagentSodium phosphate dibasic heptahydrateAcros Organics
ReagentPotassium phosphate monobasicAcros Organics
ReagentSodium phosphate dibasic heptahydrateAcros Organics
ReagentSodium chlorideMerck MilliporeSigma (Sigma-Aldrich)
ReagentPotassium phosphate monobasicAcros Organics
ReagentSodium hydroxideCatalog #795429
ReagentHydrochloric acidFisher Scientific
Safety warnings
Always put on the right Personal Protective Equipment to minimise or eliminate chances of accidental spill or splashes on the body or in the eye.
Before start
Make sure all salts and equipment to be used are available in the right quantities and functional respectively.
Preparing reagents and workspace
Preparing reagents and workspace
5m
5m
  • Before beginning the procedure put on Personal Protective Equipment (Lab Gown, Gloves, shoes, masks and goggles).

  • Clean up work surfaces and environment using disinfection solution (Bleach and after 70% alcohol).

  • Check to be sure all reagents and equipment needed for the procedure are available, in place and functional.

Weighing and dissolving salts
Weighing and dissolving salts
6m
6m
Use an electronic balance to accurately weigh out all salt powders ReagentSodium chlorideSigma Aldrich , ReagentPotassium phosphate monobasicSigma Aldrich and ReagentSodium phosphate dibasic heptahydrateSigma Aldrich into an appropriate size weighing boat with into an appropriate size weighing boat with the help of a spatula in recommended amounts as indicated in the table below.

Composition of 1X PBS buffer


ABCD
SaltConcentration (mmol/L)Concentration (g/L)Amount for 500 ml
NaCl1378.04.0
Na2HPO4101.420.71
KH2PO41.80.240.12
  • Transfer the contents of the weighing boat into a 500ml or larger beaker.
  • Use a 100ml measuring cylinder to measure 100ml of sterile distilled water and pour carefully to rinse the boat so as to take out all the powder into the beaker.

  • Mix gently by swirling and take the beaker onto a magnetic stirrer and put the magnetic flea for about Duration00:03:00 to completely dissolve the salts.
3m
Use a pH meter to measure the pH of the solution and either add ReagentSodium hydroxideSigma AldrichCatalog #795429 or ReagentHydrochloric acidSigma Aldrich to adjust the to Ph7.2 or Ph7.4 as desired.
  • Measure out the appropriate amount of sterile distilled water to make up the volume of the solution to 500ml of 1x PBS.
Critical
Sterilisation and storage
Sterilisation and storage
20m
20m
Transfer the contents of the beaker into a 500ml or 1000ml Duran Bottle and autoclave to make the PBS solution sterile.
Remove the 1x PBS from the autoclave, allow it to cool, cork tightly and store at TemperatureRoom temperature or in the refrigerator and store at Temperature4 °C . The 1x PBS buffer is ready for use in any molecular biology technique.