The first day
Preparation of CHO cells

Trypsin Solution (Nacalai)
MEM medium (GIBCO BRL)
Fetal Bovine Serum
Streptmycin/Penicillin

1. Wash CHO cells with PBS 4 ml ; 1 time
   
2. Add 2.5 ml of Trypsin solution into the above 60 mm Dish
   
3. Incubate for 10 min 37℃
   
4. Recovery the solution into 10 ml Falcon tube
   
5. Add 2.5 ml of MEM medium containing 10%FBS and antibiotics
   
6. Centrifuge for 2 min at 1,000 rpm
   
7. Discard the supernatant
   
8. Suspend the pellet with 3 ml of MEM medium containing 10%FBS and antibiotics in the Falcon tube
   
9. Add 4 ml of MEM medium containing 10%FBS and antibiotics into new 60 mm dish ( 2- 10 dishes).
   
10. Add 100 -300 μl of MEM medium suspending CHO cells from Falcon tube into each 60 mm dish containing 4 ml of MEM medium containing 10% FBS and antibiotics.
    

1. Sample (Example)
   pCMV-pChAT,　　　　0.5 μg/μl
   
2. Mix in 1.5 ml A/C Eppen tube
   

   Sample 1.0 μg is solved in 30 μl of serum free medium.
   		Sample	MEM
   PCMV-pChAT	0.5 μg/μl	2 μl x 2	28 μl x 2
   Negative control		0 μl	30 μl x 2

3. Vortex and spin
   
4. Add 4 μl of Superfect into each 1.5 ml tube and mix with pipeting
   
5. Incubate for 10 min at 25℃
   
6. Add 200 μl of MEM containing 10% serum and antibiotics
   
7. Wash wells with 1 ml of PBS
   
8. Add 234 μl of the mixture into each tube
   
9. Incubate for 2 h at 37℃ in a CO2 incubator
   
10. Wash with 1 ml of PBS
    
11. Add 1 ml of MEM containing 10% serum and antibiotics
    
12. Inucbate at 37℃ in the incubator for 24 - 48 hours