Phosphate Buffer Preparation

From LPDwiki

Necessary Materials

1. Monobasic sodium phosphate powder, (NaH2PO4)

  • MW = 137.99 g/mol

2. Dibasic sodium phosphate powder, (HNa2O4P)

  • MW = 268.07 g/mol

3. Nuclease Free water

  • Ambion Life Technologies Corporation, Product Number: AM9939

4. 15 ml tubes (x3)

Procedure

Important:

  • It is not recommended to make stock solutions that are higher than 1 M in concentration, because these require more solute, making it very hard to dissolve. As described below, 1 M stock solutions already require additional steps to dissolve.
  • The following formula was used to find required monobasic/dibasic powder weight to make 1 M stock solutions with the final volume of 10 ml:
1 M * 10 ml * MW [g/mol] = mass [g]

1 M Monobasic Phosphate Solution

1. Weigh 1.37 g of Monobasic Phosphate powder.

  • Weight found using the formula above

2. Transfer the powder to 15 ml tube.

3. Add 10 ml of nuclease free water.

4. Vortex until all powder is dissolved.

5. Label: 1 M Monobasic Sodium Phosphate in nuclease free water. (Initials, Date)

1 M Dibasic Phosphate Solution

1. Weigh 2.68 g of Dibasic Phosphate powder.

  • Weight found using the formula above

2. Transfer the powder to 15 ml tube.

3. Add 10 ml of nuclease free water.

4. Set the water bath to 60°C and let it heat up for 30 minutes.

  • Make sure that the water bath is filled with enough water to immerse a tube rack.

5. Place the 15 ml tube in the tube rack in the water bath for 30 - 60 minutes.

  • Check every 10 minutes and try to vortex at high each time.
  • The power should dissolve with vortexing and heating in 1 hour or less.Vortex until all powder dissolved.

6. Label: 1 M Dibasic Sodium Phosphate in nuclease free water. (Initials, Date)

25 mM Phosphate Buffer Solution, pH = 7.4

1. Preparing 1 M Phosphate Buffer (PB) stock

  • Mix 8.1 ml of 1 M Dibasic and 1.9 ml of 1M Monobasic Sodium Phosphate solutions in another 15 ml tube.

2. Preparing 25 mM Phosphate Buffer Solution, pH = 7.4, V = 200 ml

  • 1 M (PB stock concentration) * x (Volume of PB stock) = 25 mM (final PB concentration) * 200 ml (final 25 mM PB volume)
  • Therefore, x (Volume of PB stock) = 5 ml of 1 M PB stock
  • 5 ml of 1 M PB stock + 195 ml nuclease free DI water = 25 mM PB, pH 7.4

3. Sources for 1 M PB preparation and general procedures can be found at the following links