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

Dilute (reagent lipofectamin) 2ul with serum free mediun100ul

Dilute DNA1ul (1ug)into serum free medium 100ul

Mix DNA with lipofetamine

Incubate room temp. 15 min

Add complex to cells

37OC,4 hrs

Replace with fresh culture medium/FBS/PS( 3ml)

Add 1ml serum free medium to washed cells

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