DNA transfection. Specific methods used For Transfection 1. Electroporation a brief change of electric pulse discharges across the electrode, transiently.

Slides:



Advertisements
Similar presentations
ViahanceTM: Dead Cell, Stripped Nuclei and Free Oligonucleotide Removal Kit Instructions ViahanceTM dead cell removal kit enhances the viability ratio.
Advertisements

Immunohistochemistry ESE3 Immunohistochemistry. stained prostate tissue samples for ESE3 troubleshooted ESE3 antibody using the controls - no antibodies.
G-Fectin TM 1. A novel transfection reagent developed by Genolution 2.Cationic lipid based 3. no cell splitting is required 4. Whole transfection can be.
The Role of Innate Immune Responses in the Outcome of Interspecies Competition for Colonization of Mucosal Surfaces Lysenko ES.et al PLoS Pathog
Cat # SL Store at 4 0 C GenJet Plus DNA In Vitro Transfection Reagent A Protocol for Transfections of Mammalian Cell 100 l 500 l 1000 l.
The Elongated First Fibronectin TypeIII Domain of Collagen XIV Is An Inducer Of Quiescence And Differentiation In Fibroblasts And Preadipocytes Ruehl et.al.,(2005)
Visualizing the localization of protein isoforms in HeLa cells with laser confocal microscopy Justin R. Siebert Nancy J. Bachman, Ph.D. Biology Department.
实验 12 除去 RNA 中的基因组 DNA DNase I ( RNase Free ) TaKaRa Code : D 在微量离心管中配制下列反应液,全量 50 μl 。 全 RNA 20 ~ 50 μg 10×DNase I Buffer 5 μl DNase I ( RNase-free.
Use of Selective Media for Isolation and Enumeration of Soil Microbes 以選擇性培養基分離及計數土壤微生物.
2-D Electrophoresis 二維電泳操作教學
DNA 的抽取 元智大學通識中心 康世旭. 細胞的內容物 水 蛋白質 核酸( DNA 與 RNA) 醣類 礦物質與維生素(少量)
The construction of cells DNA or RNA (dNTP or NTP) Protein (amino acid) Carbohydrates ( 單醣 雙醣 多醣 ) Lipid etc. ( 生物膜 )
實驗十一 有機酸在水與有機溶劑間之分佈.
Dehydrogenase Activity of Soil Microbial activity in soil 以去氫脢測量土壤微生物活性.
The construction of cells
Week 5 Wednesday: –Ligation of chromosomal digests into plasmid vectors – part II – Checking ligation success Thursday: –Electrocompetent cell preparation.
Cat # SL Store at 4 0 C GenJet Plus In Vitro DNA Transfection Reagent A Protocol for generation of Lentivirus from 293T cell 100 l 500 l.
Cat # SL HEPG2 Store at 4 0 C GenJet  In Vitro DNA Transfection Reagent for HepG2 Cells (Ver. II) A Protocol for Transfecting HepG2 Cells.
BIOLOGY 3020 Fall 2008 “Keys of Corn” Project Plasmid isolation Genetic Diversity in corn. Lots of different types of corn are offered for sale at the.
Cat # SL Store at 4 0 C LipoJet DNA In Vitro Transfection Reagent A Protocol for Transfections of Mammalian Cell 100 l 500 l 1000 l
Basic technique Training and and Practice  Pipetting and transfer of fluid  Observation of cultured cells  Aseptic technique: preparation of mediums.
Cat # SL Store at 4 0 C GenJet In Vitro DNA Transfection Reagent A Protocol for Transfections of Bacmids Into Sf9 Cells 100 l 500 l 1000.
Experiment of cytobiology. schedual cell culture and counting cell isolation of subcellular structure cell proliferation assays methods in apoptosis.
Cat # SL Store at 4 0 C GenJet In Vitro DNA Transfection Reagent A Protocol for Transfections of Mammalian Cell 100 l 500 l 1000 l
实验六 动植物染色体标本的制备与 观察. 一、实验目的 学习动植物染色体标本的制备技术,掌握染色体 计数和分析的方法。 二、实验用品 1 .材料:洋葱根尖、小鼠骨髓 2 .试剂: Carnoy 固定液; 0.01 %秋水仙素;盐 酸酒精离析液; 1% 醋酸洋红染液; Giemsa 染液; 0.4%KCl.
Cat # SL Store at 4 0 C PolyJet In Vitro DNA Transfection Reagent A Protocol for Transfections of Mammalian Cell 100 l 500 l 1000 l
Cat # SL CHO Store at 4 0 C GenJet In Vitro DNA Transfection Reagent for CHO Cells (Ver. II) A Protocol for Transfections of CHO Cells 100.
Practical Part Microscopic Examination of Microorganisms Experiments Identification of MOs Different Staining Techniques.
To perform in situ studies on mRNA or DNA, cells morphology and genetic information has to be preserved. Fixation(preservation) is conducted with 3.4.
Cat # SL Store at 4 0 C SL SuperFection™ DNA Transfection Reagent Small 0.5 ml Large 1.0 ml Gaither Drive Gaithersburg, MD FAX.
Pre Lab Definitions: (Fill these in on your lab paper) Serial: In a series, order or interval. Measured steps. Dilution: Water Down. Pipette: “Little pipe”
Cat # SL Store at 4 0 C LipoD293 In Vitro DNA Transfection Reagent (Ver. II) A Protocol for generation of Lentivirus from 293T cell 100 l.
Cat # SL Store at 4 0 C GenJet DNA In Vitro Transfection Reagent 100 l 500 l 1000 l Gaither Drive Gaithersburg, MD FAX
Cell Culture Techniques
MTT ASSAY Induction of Cell Proliferation by ConA.
Bio Project 酶.
Antibody Array Assay Report 1. Protocol 2 Protein Extraction 1.Wash the cells with ice cold 1X PBS. 2.Add Lysis Beads and Extraction Buffer to the sample.
▶ Objective : To know methods that make new cell line through stable transfection 김준섭.
Cell Culture Techniques 程洪强,方瑜,孙明姣 2013 年 12 月. Cell Thawing and Transfection.
Essential Laboratory Equipment for Cell Culture Dr. Khalid Enan DFG-Sponsored Workshop, Assuit University, Egypt 10 Sep 2012.
Cat # SL Store at 4 0 C LipoD293 DNA In Vitro Transfection Reagent (Ver. II) 100  l 500  l 1000  l Gaither Drive Gaithersburg, MD
Manual Extraction of DNA from The Blood. - Blood Sample. - Distilled water. Dionized water. - Ice and Plastic bucket.- Materials.
Preparation of Metaphase Chromosomes from culturing cells.
Protocol Electro-transformation : Select a snigle colony of E.coli from fresh LB plate and inoculate to 10 ml LB broth medium.Incubate until to reach.
Add 20uL Opsonization Buffer (OB) to shaded wells 1.
Cat # SL Store at 4 0 C GenJet™ siRNA Transfection Reagent Small 0.5 ml Large 1.0 ml Gaither Drive Gaithersburg, MD FAX
Laboratory: Bacterial Transformation Introduction of plasmid DNA into E. coli E. coli.
Laboratory: Bacterial Transformation
Combined Effect of Heat, Essential Oils and Salt on the Fungicidal Activity against Trichophyton mentagrophytes in Foot Bath Shigeharu Inouye Katsuhisa.
Transfection Optimisation Process Each new siRNA screen entering the TDI requires a full transfection optimisation process. For RNAi screens, establishing.
Total bacterial number is an important indication for food quality
Supplementary figure 1A matrigel cell medium cells growing as spheroids in 3D cultures.
Cat # SL Store at 4 0 C GenJet In Vitro DNA Transfection Reagent A Protocol for Transfections of Mammalian Cell 100 l 500 l 1000 l
Products > 3T3-L1 Transfection Reagent (Embryonic Fibroblast Cells, CL-173) Altogen Biosystems offers the 3T3-L1 Transfection Reagent among a host of 100+
DNA extraction. Total DNA: whole blood (fresh or frozen), plasma, serum, buffy coat, body fluids, lymphocytes and cultured cells. This technology first.
Products > bEnd-3 Transfection Reagent (Brain Endothelioma)
Transformation of Bacteria
Products > NCI-H1299 Transfection Reagent (Lung Adenocarcinoma)
Transfection Reagent GenJet™ In vitro siRNA Small 0.5 ml Large 1.0 ml
Products > SK-MEL-28 Transfection Reagent (Melanoma Cells, HTB72)
Protocol of preparing Adhesion Assay imaging
miRNA transfection using oligofectamine
Altogen labs Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products.
Products > HCAEC Transfection Reagent (Coronary Artery Endothelial)
Mammalian Cell l 500 l l Gaither Drive Gaithersburg, MD 20877
Products > A375 Transfection Reagent (Melanoma Cells, CRL-1619)
Products > Hep-3B Transfection Reagent (Hepatocellular Carcinoma)
Products > NCI-H358 Transfection Reagent (Bronchioalveolar Cells)
Products > Transfection Reagent for Chromaffin Cells
GenJet In Vitro DNA Transfection Reagent for Hela Cells (Ver
Presentation transcript:

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 10 5 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 CO 2 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 37 O C,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 37 o C, 5 min 5.Rinse with PBS 3x(1ml/time) 6.Add 0.5 ml x-Gal stock buffer 7.Stain 37 o C, 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 Cl 2 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( 5x10 6 cells) 2.cell harvest, 置於 15 ml 離心管 rpm 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. 37 o C, 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