TITLE: Preparation of PBS Solution
AUTHORS: Stephane  Fadanka, Shalo  Minette, Nadine          Mowoh

[DESCRIPTION]
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. 




[BEFORE_START]
Make sure all salts and equipment to be used are available in the right quantities and functional respectively.

[GUIDELINES]
This protocol can be performed by anyone with basic lab skills

[STEPS]
SECTION: Preparing reagents and workspace
1. 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.
 

SECTION: Weighing and dissolving salts
2. Use an electronic balance to accurately weigh out all salt powders  ,   and    into an appropriate size weighing boat with the help of a spatula as indicated in the table below.

Composition of 1X PBS buffer
 
    SaltConcentration (mmol/L)Concentration (g/L)Amount for 500 mlNaCl1378.04.0Na2HPO4101.420.71KH2PO41.80.240.12
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 3 min on a magnetic stirrer to rinse the boat to take out any powder residue' completely dissolve the salts.
SECTION: Weighing and dissolving salts
3. Check the pH of the solution and either add 1M  or 3 M  gradually to adjust the to pH 7.2or pH 7.4as 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.

SECTION: Sterilisation and storage
4. Transfer the contents of the beaker into a 1000 mL Duran bottle and autoclave to make the PBS solution sterile. 
SECTION: Sterilisation and storage
5. Remove the 1x PBS from the autoclave, allow it to cool, cork tightly and store at 23 Room temperature or in the refrigerator and store at 4 °C. The 1x PBS buffer is ready for use in any molecular biology technique.