DNA transfection
Specific methods used For Transfection
1. Electroporation
 a brief change of electric pulse discharges
across the electrode, transiently open holes
in cells
2. Liposomediated gene transfer
 liposome fuse directly with cell membrane
and delivers DNA into cells
DNA transfection by lipofectamine
Procedure:
1.Trypsinize a confluent cells as previously described.
2.Plate cell approximately 105 cells/ 24well dish with
3. Incubate cells till 50-70% of confluency( 18-24hrs before
transfection)
4. Wash cells with PBS for 3 times( 1ml each), and add 0.4 ml
of serum free- medium to the cells
5.In an eppendorf tube, pipette 1ug of DNA, 1ul of
lipofectamine 2000 and 100 ul of serum free medium
6. Incubate at room temperature for 15 min to allow the DNA
lipofetamine complex to form
7. Add DNA- lipofectamine complex to the cells drop wise
while swirling the dish.
8. Incubate cells in an CO2 incubator for at least 3hrs
9. Remove transfection medium and replace with 1 ml of
culture medium
10.Analyze cell 24-48hrs for ß-galactosidase activity
Experimental Pocedure For DNATransfection
Plate cells 1 day before
transfection
cells wash with PBS
3x(1ml/time) before
transfection
Add 1ml
serum free
medium to
washed cells
Dilute (reagent
lipofectamin) 2ul
with serum free
mediun100ul
Mix DNA with lipofetamine
Incubate room temp. 15 min
Add complex to cells
37OC,4 hrs
Replace with fresh culture
medium/FBS/PS( 3ml)
Dilute DNA1ul
(1ug)into serum
free medium 100ul
X-Gal stainning
1.remove culture medium
2.cells wash with PBS 3x(1ml/time)
3.fix with 0.5% glutaldehyde/PBS
4.incubate 37oC, 5 min
5.Rinse with PBS 3x(1ml/time)
6.Add 0.5 ml x-Gal stock buffer
7.Stain 37oC, 4 hrs
8.Rinse with PBS 2x(1ml/time)
9.Count blue cell under inverted microscope and
calculate the efficiency of transfection
X-gal ( 5-bromo-4chloro-3-indolyl-ß-D galactoside)stock buffer
3 mM KeFe( CN)6
3 mM K4 Fe( CN)6
1mM Mg Cl2
10 mM KCl
0.1% Triton X-100
Dilute x-gal 1:100 in X-gal stock buffer
Electroporation
1.Plate cell in 10 mm dish( 5x106 cells)
2.cell harvest,置於15 ml 離心管
3.1200rpm 離心三分鐘
4.倒去上清液
5.將細胞回溶於0.5 ml SF( serum free) medium
6.將細胞放入電擊管
7.細胞通電
8.將細胞取出, 放入有蓋玻片, 3ml 培養基之6mm 培養皿
9. 37oC , 24 hrs
10. 取出細胞( 到 931 lab)
11. 以 PBS 清洗2 次( 1ml each)
12. 加入4 % paraformaldehyde/PBS,靜置室溫三十分鐘
12. 加入 DAPI溶液,靜置室溫五分鐘並避光
13. 移除DAPI溶液
14. 以 PBS 清洗3 次( 1ml each)
15. 滴一滴PBS 於載玻片
16.取出蓋玻片, 倒蓋於在載玻片上
17. 封片
18. 以螢光顯微鏡觀察