Jul 28, 2022
Version 2
Preparation of PBS Solution V.2
- 1Mboalab, Beneficial Bio
Protocol Citation: Stephane Fadanka, Mujar Minette Shalo, Nadine Mowoh 2022. Preparation of PBS Solution. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4bxjrvo5/v2Version created by Nadine Mowoh
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: July 28, 2022
Last Modified: July 28, 2022
Protocol Integer ID: 67831
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.
Guidelines
This protocol can be performed by anyone with basic lab skills
Materials
Reagents
- Sodium chlorideSigma – Aldrich
- Sodium phosphate dibasic heptahydrateAcros Organics
- Potassium phosphate monobasicAcros Organics
- Sodium hydroxide
- Hydrochloric acidFisher Scientific
Materials and equipment
- 500 mL Beaker
- 100 mL Measuring Cylinder
- Distilled water
- pH Meter
- Electronic Balance
- Weighing boat
- Magnetic Stirrer
- Refrigerator
- Autoclave
- 1000 mL Duran Bottle
Protocol materials
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)
Step 2
Sodium phosphate dibasic heptahydrateAcros Organics
Step 2
Sodium hydroxideCatalog #795429
Step 3
Sodium chlorideMerck MilliporeSigma (Sigma-Aldrich)
Materials
Sodium phosphate dibasic heptahydrateAcros Organics
Materials
Sodium hydroxide
Materials
Hydrochloric acidFisher Scientific
Materials, Step 3
Potassium phosphate monobasicAcros Organics
Materials
Potassium phosphate monobasicAcros Organics
Step 2
Safety warnings
- Always put on the right Personal Protective Equipment to minimize or eliminate chances of accidental spill or splashes on the body or in the eye.
- Special caution should be taken while handling HCL because it it corrosive
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
- 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).
- Ensure that all reagents and equipment needed for the procedure are available, in place and functional.
Preparing 1 M NaOH
- Weigh 19.95 g of NaOH pellets & dissolve them in half liter (500 mL) of distilled water water.
Preparing 3 M HCL
- Measure out 8.5 mL of distilled water into a cleaned and dried 50 mL volumetric flask. Add 2.5 mL of Concentrated HCL.
Safety information
Remember to add acid to water, the volumetric flask should be part filled with water before adding the acid.
10m
Weighing and dissolving salts
Weighing and dissolving salts
6m
6m
Use an electronic balance to accurately weigh out all salt powders Sodium chlorideSigma Aldrich , Potassium phosphate monobasicSigma Aldrich and Sodium phosphate dibasic heptahydrateSigma Aldrich into an appropriate size weighing boat with the help of a spatula as indicated in the table below.
Composition of 1X PBS buffer
| A | B | C | D | |
| Salt | Concentration (mmol/L) | Concentration (g/L) | Amount in grams (g) for 500 mL | |
| NaCl | 137 | 8.0 | 4.0 | |
| Na2HPO4 | 10 | 1.42 | 0.71 | |
| KH2PO4 | 1.8 | 0.24 | 0.12 |
- Transfer the contents of the weighing boat into a 500 mL or larger beaker.
- Use a 100ml measuring cylinder to measure out 100 mL of sterile distilled water and pour carefully into the beaker and rinse the boat to take out all the powder residue.
- Mix gently by swirling. Use a magnetic flea to stir the solution for 00:03:00 on a magnetic stirrer to rinse the boat to take out any powder residue' completely dissolve the salts.
3m
Check the pH of the solution and either add 1M Sodium hydroxideSigma AldrichCatalog #795429 or 3 MHydrochloric acidSigma Aldrich gradually to adjust the to 7.2 or 7.4 as desired.
- After confirming the pH, measure out the appropriate amount of sterile distilled water to make up the volume of the solution to 500 mL of 1x PBS.
5m
Sterilisation and storage
Sterilisation and storage
Transfer the contents of the beaker into a 1000 mL Duran bottle and autoclave to make the PBS solution sterile.
30m
Remove the 1x PBS from the autoclave, allow it to cool, cork tightly and store at Room temperature or in the refrigerator and store at 4 °C . The 1x PBS buffer is ready for use in any molecular biology technique.