How to Make Phosphate Buffered Saline
Gather your materials and ingredients., Place the stir bar in the beaker and place it on top of stir plate., Measure out 800 mL of distilled water into the 2 L beaker., Weigh out the proper amounts of each chemical., Add all of the chemicals to the...
Step-by-Step Guide
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Step 1: Gather your materials and ingredients.
To make a 1X (the normal concentration used for experiments) solution of phosphate buffered saline (PBS), you will need a 1 L graduated cylinder, a balance, a 2 L beaker, stir bar, stir plate, pH meter, autoclave, and a glass bottle for storage.
To make 1 L, you will need the following amounts of each ingredient:
You will need small amounts of dilute HCl and/or NaOH to pH the final solution. start with 800 mL of distilled water 8 g of NaCl (sodium chloride)
0.2 g of KCl (potassium chloride)
1.44 g of Na2HPO4 (disodium phosphate)
0.24 g of KH2PO4 (monopotassium phosphate) The final concentrations of each compound should be 137 mm NaCl, 10 mM Na2HPO4,
1.8 mM KH2PO4,
2.7 mM KCl, and a pH
7.4. -
Step 2: Place the stir bar in the beaker and place it on top of stir plate.
When making any solution, you want to keep the solution constantly moving.
This will help everything dissolve faster.
To avoid anything splashing, place the stir bar into the empty beaker and then put it on top of the stir plate.Wait until there is solution in the beaker before turning it on. , When preparing a solution, start with less than the final volume of water.
At the end, you will add the rest of the water to bring the total volume up to 1 L.
Using the graduated cylinder, measure 800 mL of water and pour this directly into a clean beaker.With the water in the beaker, turn on the stir plate to a medium speed.
You should see a small whirlpool form in the water as the stir bar begins to spin. , Weigh out 8 g of NaCl,
0.2 g of KCl,
1.44 g of Na2HPO4 and
0.24 g of KH2PO4.
Use the balance to weigh out each chemical one at a time in their own weigh boats.If you add too much chemical, simply remove the excess until you have the right amount. , Pour each chemical into the water while it is stirring.
There is no special order to add each one, so you can add everything all at once.
Let everything continue to stir until it is fully dissolved.You can let this sit while you do something else and check on it later.
It will take at least 5-10 minutes to dissolve. , Once everything has fully dissolved, you need to adjust the pH of the buffer until it is
7.4.Keep the stir bar on and place the pH meter into the top of the solution.
Let the numbers on the pH meter stop moving.
The solution will likely be too acidic so you’ll need to add dilute sodium hydroxide (NaOH) to adjust.
Use a small pipette to add NaOH a few drops at a time.
After each addition, let the solution stir for a moment and check the new reading on the pH meter.
To pH a solution is a process that requires patience.
If you accidentally go higher than
7.4, you can add a few drops of hydrochloric acid (HCl) to bring it back down. , After the pH has been properly adjusted, you can bring your solution up to the final volume of 1 liter.
Carefully pour the entire solution from the beaker into the 1 L graduated cylinder.
Add enough distilled water to make it 1 L.Pour the final solution into a 1 L glass bottle safe for autoclaving. , Place the lid on the bottle, but do not screw it down.
During autoclaving, the pressure in the bottle can build up and explode if you tighten the lid.
Put a strip of autoclave tape across the lid to hold it in place while autoclaving.Set the autoclave to sterilize for 20 minutes at 15 psi on the liquid cycle.Autoclave tape has a temperature indicator in it.
If the autoclave tape doesn’t turn black when the process is done, the autoclave did not get hot enough and you will need to re-sterilize. , After autoclaving, let the PBS cool to room temperature and then tighten the lid to keep the contaminants out.
Tightening the lid before the bottle cools can create a vacuum and make it very difficult to remove the lid later.
PBS is stable at room temperature.You can also aliquot the PBS into smaller containers to use one at a time. -
Step 3: Measure out 800 mL of distilled water into the 2 L beaker.
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Step 4: Weigh out the proper amounts of each chemical.
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Step 5: Add all of the chemicals to the solution and dissolve.
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Step 6: Adjust the pH of the solution to 7.4.
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Step 7: Top off the water until the solution is 1 liter.
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Step 8: Sterilize in an autoclave.
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Step 9: Store PBS at room temperature.
Detailed Guide
To make a 1X (the normal concentration used for experiments) solution of phosphate buffered saline (PBS), you will need a 1 L graduated cylinder, a balance, a 2 L beaker, stir bar, stir plate, pH meter, autoclave, and a glass bottle for storage.
To make 1 L, you will need the following amounts of each ingredient:
You will need small amounts of dilute HCl and/or NaOH to pH the final solution. start with 800 mL of distilled water 8 g of NaCl (sodium chloride)
0.2 g of KCl (potassium chloride)
1.44 g of Na2HPO4 (disodium phosphate)
0.24 g of KH2PO4 (monopotassium phosphate) The final concentrations of each compound should be 137 mm NaCl, 10 mM Na2HPO4,
1.8 mM KH2PO4,
2.7 mM KCl, and a pH
7.4.
When making any solution, you want to keep the solution constantly moving.
This will help everything dissolve faster.
To avoid anything splashing, place the stir bar into the empty beaker and then put it on top of the stir plate.Wait until there is solution in the beaker before turning it on. , When preparing a solution, start with less than the final volume of water.
At the end, you will add the rest of the water to bring the total volume up to 1 L.
Using the graduated cylinder, measure 800 mL of water and pour this directly into a clean beaker.With the water in the beaker, turn on the stir plate to a medium speed.
You should see a small whirlpool form in the water as the stir bar begins to spin. , Weigh out 8 g of NaCl,
0.2 g of KCl,
1.44 g of Na2HPO4 and
0.24 g of KH2PO4.
Use the balance to weigh out each chemical one at a time in their own weigh boats.If you add too much chemical, simply remove the excess until you have the right amount. , Pour each chemical into the water while it is stirring.
There is no special order to add each one, so you can add everything all at once.
Let everything continue to stir until it is fully dissolved.You can let this sit while you do something else and check on it later.
It will take at least 5-10 minutes to dissolve. , Once everything has fully dissolved, you need to adjust the pH of the buffer until it is
7.4.Keep the stir bar on and place the pH meter into the top of the solution.
Let the numbers on the pH meter stop moving.
The solution will likely be too acidic so you’ll need to add dilute sodium hydroxide (NaOH) to adjust.
Use a small pipette to add NaOH a few drops at a time.
After each addition, let the solution stir for a moment and check the new reading on the pH meter.
To pH a solution is a process that requires patience.
If you accidentally go higher than
7.4, you can add a few drops of hydrochloric acid (HCl) to bring it back down. , After the pH has been properly adjusted, you can bring your solution up to the final volume of 1 liter.
Carefully pour the entire solution from the beaker into the 1 L graduated cylinder.
Add enough distilled water to make it 1 L.Pour the final solution into a 1 L glass bottle safe for autoclaving. , Place the lid on the bottle, but do not screw it down.
During autoclaving, the pressure in the bottle can build up and explode if you tighten the lid.
Put a strip of autoclave tape across the lid to hold it in place while autoclaving.Set the autoclave to sterilize for 20 minutes at 15 psi on the liquid cycle.Autoclave tape has a temperature indicator in it.
If the autoclave tape doesn’t turn black when the process is done, the autoclave did not get hot enough and you will need to re-sterilize. , After autoclaving, let the PBS cool to room temperature and then tighten the lid to keep the contaminants out.
Tightening the lid before the bottle cools can create a vacuum and make it very difficult to remove the lid later.
PBS is stable at room temperature.You can also aliquot the PBS into smaller containers to use one at a time.
About the Author
Carol Parker
Creates helpful guides on home improvement to inspire and educate readers.
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