DNA transfection

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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
DNA transfectionSpecific methods used For Transfection1. Electroporation a brief change of electric pulse discharges across the electrode, transiently open holes in cells2. Liposomediated gene transfer liposome fuse directly with cell membrane and delivers DNA into cellsDNA transfection by lipofectamineProcedure: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 cells5.In an eppendorf tube, pipette 1ug of DNA, 1ul of lipofectamine 2000 and 100 ul of serum free medium6. Incubate at room temperature for 15 min to allow the DNA lipofetamine complex to form7. Add DNA- lipofectamine complex to the cells drop wise while swirling the dish.8. Incubate cells in an CO2 incubator for at least 3hrs9. Remove transfection medium and replace with 1 ml of culture medium10.Analyze cell 24-48hrs for ß-galactosidase activityExperimental Pocedure For DNATransfectionPlate cells 1 day before transfection cells wash with PBS 3x(1ml/time) before transfectionDilute (reagent lipofectamin) 2ul with serum free mediun100ulDilute DNA1ul (1ug)into serum free medium 100ulAdd 1ml serum free medium to washed cellsMix DNA with lipofetamineIncubate room temp. 15 minAdd complex to cells37OC,4 hrsReplace with fresh culture medium/FBS/PS( 3ml)X-Gal stainning1.remove culture medium2.cells wash with PBS 3x(1ml/time)3.fix with 0.5% glutaldehyde/PBS4.incubate 37oC, 5 min5.Rinse with PBS 3x(1ml/time)6.Add 0.5 ml x-Gal stock buffer7.Stain 37oC, 4 hrs8.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-100Dilute x-gal 1:100 in X-gal stock bufferElectroporation1.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) medium6.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 hrs10. 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
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