Technical Bulletin TransFast TM Transfection Reagent E /
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1 Technical Bulletin TransFast TM Transfection Reagent E /
2 TransFast TM Transfection Reagent E2431 I II III A. DNA...2 B. DNA...3 C....3 D....3 E....3 IV A....3 B....4 C....5 D....6 E....7 F....8 V VII I. 1DEAE(1,2) (3) (3)(4,5) L-dioleoyl phosphatidylethanolamine (DOPE) TransFast TM Transfection Reagent(+)-N,N [bis (2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide(1) DOPETransFast TM Transfection Reagent (7,8) / (9) (10)DOPE (10)RNA DNA/DNA TEL: , prometec@jp.promega.com 1
3 1 TransFast TM TransFast TM in vitroin vivo RNA DNA (11-16)TransFast TM Transfection Reagentin vitroin vivo(17) DNA NIH/3T3CHO293K562PC12JurkatSf9 TransFast TM Transfection Reagent II. TransFast TM Transfection Reagent 1.2mg E2431 TransFast TM Transfection ReagentDNA11400µgDNA 1.2mg TransFast TM Transfection Reagent (3 0.4mg) 2ml Nuclease-Free Water ()TransFast TM Transfection Reagent C 6Nuclease Free Water TransFast TM Transfection Reagent C8 TransFast TM Transfection Reagent Nuclease-Free Water III. DNA Transfection Assistant Transfection Assistant: III.A. DNA in vitrodna DNA DNA Section ITransFast TM ReagentDNA 1:12:1 TEL: , prometec@jp.promega.com 2
4 III.B. DNA DNARNA (A260:A280=1.81.9)DNATE Buffer 0.21mg/mlDNA DNA DNA2:11: µgDNADNA III.C. DNA TransFast TM Reagent30 III.D. TransFast TM Reagent III.E. TransFast TM Reagent IV. 23 ( ) 24660mm100mm IV.A. 80% HeLa mm TEL: , prometec@jp.promega.com 3
5 1TransFast TM Nuclease Free-Water -20 2DNA TransFast TM DNA DNATransFast TM (1) 37 (48) 1 1(cm 2 ) a b 24-well x 96-well 12-well 6-well 35mm 60mm x 100mm x a: Corning b: 24well-plate IV.B. 1. TransFast TM Reagent400µl Nuclease-Free Water(1mM) Nuclease-Free Water10 20 C 2 x 5 x 4.2 x Note: 2. 50ml300xg TransFast TM Reagent 20 C(TransFast TM Reagent 10) Note: TransFast TM ReagentNuclease-Free Water 11 x TEL: , prometec@jp.promega.com 4
6 TransFast TM Reagent3730 2TransFast TM Reagent IV.C. 23 (CAT)β- (VII) DNA( µg)(TransFast TM ReagentDNA) (n=6) DNATransFast TM Reagent 200µl2A2B7DNA 6 (37)DNA TransFast TM Reagent 3 24 TEL: , prometec@jp.promega.com 5
7 2A TransFast TM ReagentDNA1:1 1DNA DNA 0.25 µg 0.5 µg 0.75 µg 1.0 µg 1.8 µg 3.5 µg 5.3 µg 7.0 µg TransFast TM Reagent* 5.4 µl 10.5 µl 15.9 µl 21 µl *)TransFast TM Reagent 1400µl 7DNA6 2B TransFast TM ReagentDNA2:1 1DNA DNA 0.25 µg 0.5 µg 0.75 µg 1.0 µg 1.8 µg 3.5 µg 5.3 µg 7.0 µg TransFast TM Reagent* 10.8 µl 21 µl 31.8 µl 42 µl *)TransFast TM Reagent 1400µl 7DNA6 Note: DNA Transfection CalculatorURL 2. TransFast TM ReagentDNA TransFast TM ReagentDNA µl1( ) mlTransFast TM Reagent DNA ( CATβ-)48 6. IV.D. Note: TransFast TM Reagent() 60mm ( 100mm ) 3 TEL: , prometec@jp.promega.com 6
8 3 DNATransFast TM Reagent (1) DNA (1) 6-well 1.0 ml 1.255µg 12-well 24-well 96-well 400 µl 200 µl 40 µl µg µg µg 1. 60mm DNA TransFast TM Reagent 2ml (100mm 6ml) µg DNA (100mm 7.530µg) 60mm 5µg DNA (100mm 15µg DNA )TransFast TM Reagent DNA 11 4 TransFast TM Reagent 4 TransFast TM ReagentTransFast TM ReagentDNA 1µgDNA TransFast DNA TransFast TM Reagent* µl 6.0 µl µl *)TransFast TM Reagent 1400µl 2. TransFast TM ReagentDNA TransFast TM ReagentDNA2ml(100mm 6ml)1() ml(100mm 12ml) TransFast TM ReagentDNA IV.E. Note: TransFast TM Reagent() µgDNATransFast TM Reagent 111DNA TransFast TM ReagentDNA 1. TransFast TM Reagent g /ml 3. TransFast TM ReagentDNA370.5m TEL: , prometec@jp.promega.com 7
9 DNA5TransFast TM Reagent 5 TransFast TM ReagentDNA11 1DNA DNA 1µg 2µg 3µg 4µg 0.5ml 0.5ml 0.5ml 0.5ml DNA 1µg 2µg 3µg 4µg TransFast TM Reagent* 3µl 6µl 9µl 12µl *)TransFast TM Reagent1400µl 4. TransFast TM ReagentDNA TransFast TM ReagentDNA0.5ml( )6 6. TransFast TM ReagentDNA(0.5ml/well) 1() ml ( 48) 8. IV.F. DNA DNA 12 () 23 () G418(18) B B(19) 5790% IV.D.IV.E. TEL: , prometec@jp.promega.com 8
10 DNA ( ) ( ) 4. ()96 Note: ( 2) 2. 2%(60%) () 8. TEL: , prometec@jp.promega.com 9
11 V. DNACsCl DNAA260:A TransFast TM ReagentDNA TransFast TM ReagentDNA 1:12:1 TransFast TM Reagent DNATransFast TM Reagent TransFast TM ReagentDNA TEL: , 10
12 VI. 1. Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, McCutchan, J.H. and Pagano, J.S. (1968) Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Natl. Cancer Inst. 41, Sessa, G. and Weissmann, G. (1968) Phospholipid spherules (liposomes) as a model for biological membranes. J. Lipid Res. 9, Felgner, P.L. et al. (1987) Lipofection: A highly efficient, lipid-mediated DNA transfection procedure. Proc. Natl. Acad. Sci. USA 84, Felgner, J.H. et al. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, Wheeler, C.J. et al. (1996) Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes. Biochim. Biophys. Acta 1280, Kabanov, A.V. and Kabanov, V.A. (1995) DNA complexes with polycations for the delivery of genetic material into cells. Bioconjug. Chem. 6, Labat-Moleur, F. et al. (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 3, Gao, X. and Huang, L. (1995) Cationic liposome-mediated gene transfer. Gene Ther. 2, Farhood, H., Serbina, N. and Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta 1235, Capaccioli, S. et al. (1993) Cationic lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells and in human serum. Biochem. Biophys. Res. Comm. 197, Felgner, J., Bennet, F. and Felgner, P.L. (1993) Cationic lipid mediated delivery of polynucleotides. Methods: A Companion to Methods in Enzymology 5, Lee, J.T. and Jaenisch, R. (1996) A method for high efficiency YAC lipofection into murine embryonic stem cells. Nucl. Acids Res. 24, Lamb, B.T. and Gearhart, J.D. (1995) YAC transgenics and the study of genetics and human disease. Curr. Opin. Genet. Dev. 5, Wilson, T., Papahadjopoulos, D. and Taber, R. (1979) The introduction of poliovirus RNA into cells via lipid vesicles (liposomes). Cell 17, Malone, R.W., Felgner, P.L. and Verma, I.M. (1989) Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86, Bennett, M.J. et al. (1997) Cationic lipid-mediated gene delivery to murine lung:correlation of lipid hydration with in vivo transfection activity. J. Med. Chem. 40, Southern, P.J. and Berg, P. (1982) Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1, Blochlinger, K. and Diggelmann, H. (1984) Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Mol. Cell. Biol. 4, TEL: , prometec@jp.promega.com 11
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1 Department of Cancer and Thoracic Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan; 2 Department of Thoracic Surgery, Aichi Cancer Center, Japan;
