Preparation of MEM & PBS

Cell Culture Lab (VM3417) Procedure

Preparation of Minimum Essential Medium (MEM)

 PrepareMEM

Investigator: __________       Date: ___________    Protocol: ______________________

1. Heat-inactivate Fetal Bovine Serum (FBS), (Hyclone# HAD3016002) for 30 minutes at 56ºC. (before use).

Date of Heat-inactivate Fetal Bovine Serum (FBS)………………………………

2. Preparation of Minimum Essential Medium (MEM-EAGLE, Gibco^® # 1IVG1-61100-061) For 1 Litter.

Measure out 950 ml (in a graduated cylinder) of ultrapure water (use distilled water Deionize Water, DI water (sometime use)) (water temperature should be 15-20 °C.)  Pour into a Duran bottle.

While gently stirring (use a stir plate and stir bar) the water, add the entire package of powdered medium. Stir until dissolved.

Autoclave for 10 minutes at 115ºC. (should be at room temperature (15-20 °C.)

To the solution previously prepared, add 0.85 g (g/Litter) of powdered, cell culture grade, sodium bicarbonate (AMRESCO^® # 0865-1). Measure out  30 ml (in a graduated cylinder) of ultrapure water (use distilled water Deionize Water, DI water (sometime use)). Stir until dissolved.

Sterilize immediately by filtration using a membrane with a porosity of 0.22 microns or less.

Add the Glutamax I supplement (Gibco^® # 1IVG7-35050-061) and Sodium pyruvate solution (Gibco^® # 1IVG7-11360-070) add to 10 ml/Litter  respectively.

Store liquid medium by refrigerating at 0-5 °C.

Notes:

• Powdered medium is Minimal Essential Medium Eagle, with Non-Essential Amino Acids, Earle’s Salts and L-Glutamine, without Sodium Bicarbonate. Glutamax I supplement and Sodium pyruvate solution (Gibco^® Company).

• Refrigerate powdered medium, Glutamax I supplement  and Sodium pyruvate solution at 0-5 °C.

• Preparing a concentrated solution of media is not recommended.  Some free base amino acids have low solubility coefficients and insoluble salt complexes may precipitate in concentrated solution.

• It is helpful to heat-inactivate batches of serum sometime before making medium and store the heated serum in 15 ml aliquots at –20ºC.

• This procedure was revised on 24-06-2013 by Sunsanee Supunkong.

 

Cell Culture Lab (VM3417) Procedure

Preparation of Phosphate Buffer Saline (PBS)

 Prepare PBS

Investigator: __________       Date: ___________    Protocol: ______________________

1. A 1 liter stock of 10x PBS can be prepared by dissolving

– Add 80 g NaCl,

– Add 2 g KCl,

– Add 11.5 g Na₂HPO₄

– Add 2 g KH₂PO₄

– Add 1 Litter of Distilled water

After complete mixing, top up final solution to 1 L. The pH of the 10X stock is will be approximately 6.8, but when diluted to 1x PBS it should change to 7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide.

Sterilize by autoclaving (20 min, 121°C). Store at room temperature.

2. For 1 liter of 1X PBS, prepare as follows:

Start with 800 ml of distilled water:

– Add 8 g of NaCl.

– Add 0.2 g of KCl.

– Add 1.15 g of Na2HPO4.

– Add 0.24 g of KH2PO4.

– Adjust the pH to 7.4 with HCl. And add distilled water to a total volume of 1 liter.

Dispense the solution into aliquots and sterilize by autoclaving (20 min, 121°C). Store at room temperature.

Or used 10XPBS by add 100 ml of 10XPBS, and add 900 ml of Distilled water sterilize by autoclaving (20 min, 121°C). Store at room temperature.

Notes:

• This procedure was revised on 24-06-2013 by Sunsanee Supunkong.