Dulbeccos Modified Eagle's Media, DMEM adding extras

Required equipment

• 500 mL DMEM
• 50 mL FBS
• 5 mL Pen-Strep
• 0.2 µm filters
• 20 mL syringe
• 60 mL syringe

Protocol

1. Before prepare for DMEM solution, take out 1 tube of FBS and 1 tube pen-strep from the freezer and place them in the fridge (1 day in advance, after the overnight is fine).
2. Work inside the LAF bench and use syringes and 0.2 µm filter to add 50 ml FBS and 5 ml Pen-Strep to the 500ml DMEM.
3. Tighten the caps, and slightly shake the bottle to mix them. 
4. Label the bottle, then store it in the fridge:

   name, dd/mm/yy, +10% FBS, +1% P/S 

Stock solution preparation

It is best to prepare a few tubes with the required amount of FETAL BOVINE SERUM (FBS) and Penicilin Streptomycin (Pen-Strep), and store them in the freezer. Each time only unfreeze the small tubes, instead of the whole bottle. As for 500 ml DMEM, it requires 50 mL (or 10%) of FBS and 5 mL (or 1%) of Penicilin Streptomycin (Pen-Strep).

FETAL BOVINE SERUM (FBS)  

1. Use 75% antibac ethanol alcohol to spray both hands with gloves, then the surface of the FBS bottle, the surface of 25 ml Serological Pipette, a few of 50 ml conical tube, and place them inside the LAF fume hood.  
2. Inside the LAF hood, completely loose the conical tube caps and placed them in frame holder. Transfer 51 ml FBS into each conical tube, and tighten the caps.  
3. Label each conical tube:

    50 mL FBS, dd/mm/yy, name 

4. Store tubes in the freezer. 

Penicilin Streptomycin (Pen-Strep)

1. Use 75% antibac ethanol alcohol to spray both hands with gloves, then the surface of the FBS bottle, the surface of 25 ml Serological Pipette, a few of 15 ml conical tube, and place them inside the LAF fume hood.  
2. Inside the LAF hood, completely loose the conical tube caps and placed them in frame holder. Transfer 5.5 ml Pen-Strep into each conical tube, and tighten the caps.  
3. Label each conical tube:

    5 mL P/S, dd/mm/yy, name 

4. Store tubes in the freezer.