Technical Bulletin TransFast TM Transfection Reagent E2431 http://www.promega.com/tbs/tb260/tb260.pdf / www.promega.co.jp 2006 8
TransFast TM Transfection Reagent E2431 I.................................................................... 1 II.................................................. 2 III............................................................. 2 A. DNA...2 B. DNA...3 C....3 D....3 E....3 IV.........................................................3 A....3 B....4 C....5 D....6 E....7 F....8 V............................................................10 VII....................................................................11 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:03-3669-7980, prometec@jp.promega.com 1
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 15 25 C 6Nuclease Free Water TransFast TM Transfection Reagent 15 25 C8 TransFast TM Transfection Reagent Nuclease-Free Water III. DNA Transfection Assistant Transfection Assistant: www.promega.com/transfectionasst/ III.A. DNA in vitrodna DNA DNA Section ITransFast TM ReagentDNA 1:12:1 TEL:03-3669-7980, prometec@jp.promega.com 2
III.B. DNA DNARNA (A260:A280=1.81.9)DNATE Buffer 0.21mg/mlDNA DNA DNA2:11:12410.250.500.75 1µgDNADNA III.C. DNA 11 304 TransFast TM Reagent30 III.D. TransFast TM Reagent III.E. TransFast TM Reagent IV. 23 ( ) 24660mm100mm IV.A. 80% HeLa 24 1 510 4 60mm 5.510 5 1 TEL:03-3669-7980, prometec@jp.promega.com 3
1TransFast TM Nuclease Free-Water -20 2DNA TransFast TM DNA DNATransFast TM 1015 37 (1) 37 (48) 1 1(cm 2 ) a b 24-well 1.88 1 x 96-well 12-well 6-well 35mm 60mm 0.32 3.83 9.4 8.0 21 0.2 x 100mm 55 29 x a: Corning b: 24well-plate 510 4 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:03-3669-7980, prometec@jp.promega.com 4
TransFast TM Reagent3730 2TransFast TM Reagent IV.C. 23 (CAT)β- (VII) DNA(0.250.50.751µg)(TransFast TM ReagentDNA)112 1 1224 1(n=6)3 1. 241DNATransFast TM Reagent 200µl2A2B7DNA 6 (37)DNA TransFast TM Reagent 3 24 TEL:03-3669-7980, prometec@jp.promega.com 5
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 www.promega.com/techserv/tools/transcalc 2. TransFast TM ReagentDNA101530 3. 4. TransFast TM ReagentDNA241 200µl1( )37 5. 371mlTransFast TM Reagent DNA ( CATβ-)48 6. IV.D. Note: TransFast TM Reagent() 60mm ( 100mm ) 3 TEL:03-3669-7980, prometec@jp.promega.com 6
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 0.5 2 µg 0.251 µg 0.050.2 µg 1. 60mm DNA TransFast TM Reagent 2ml (100mm 6ml) 372.5 10µg 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* 11 21 3.0 µl 6.0 µl 31 9.0 µl *)TransFast TM Reagent 1400µl 2. TransFast TM ReagentDNA1015 3. 4. TransFast TM ReagentDNA2ml(100mm 6ml)1() 37 5. 374ml(100mm 12ml) TransFast TM ReagentDNA 6. 48 IV.E. Note: TransFast TM Reagent() 110 6 1234µgDNATransFast TM Reagent 111DNA TransFast TM ReagentDNA 1. TransFast TM Reagent-20 2. 110 6 300g5210 6 /ml 3. TransFast TM ReagentDNA370.5m TEL:03-3669-7980, prometec@jp.promega.com 7
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 ReagentDNA1015 5. TransFast TM ReagentDNA0.5ml(1 10 6 )6 6. TransFast TM ReagentDNA(0.5ml/well) 1() 37 7. 5ml ( 48) 8. IV.F. DNA DNA 12 () 23 () G418(18) B B(19) 5790% IV.D.IV.E. TEL:03-3669-7980, prometec@jp.promega.com 8
DNA 1. 48 (12011001200) 2. 1434 3. 2( ) 4. ()96 Note: 1. 14 ( 2) 2. 2%(60%) 3. 4. 5. 5 6. 7. () 8. TEL:03-3669-7980, prometec@jp.promega.com 9
V. E-mail prometec@jp.promega.com DNACsCl DNAA260:A2801.81.9 TransFast TM ReagentDNA TransFast TM ReagentDNA 1:12:1 TransFast TM Reagent DNATransFast TM Reagent TransFast TM ReagentDNA TEL:03-3669-7980, prometec@jp.promega.com 10
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, 456-67. 2. 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, 351-7. 3. Sessa, G. and Weissmann, G. (1968) Phospholipid spherules (liposomes) as a model for biological membranes. J. Lipid Res. 9, 310-8. 4. Felgner, P.L. et al. (1987) Lipofection: A highly efficient, lipid-mediated DNA transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413-7. 5. Felgner, J.H. et al. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-61. 6. 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, 1-11. 7. Kabanov, A.V. and Kabanov, V.A. (1995) DNA complexes with polycations for the delivery of genetic material into cells. Bioconjug. Chem. 6, 7-20. 8. Labat-Moleur, F. et al. (1996) An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. 3, 1010-7. 9. Gao, X. and Huang, L. (1995) Cationic liposome-mediated gene transfer. Gene Ther. 2, 710-22. 10. Farhood, H., Serbina, N. and Huang, L. (1995) The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta 1235, 289-95. 11. 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, 818-25. 12. Felgner, J., Bennet, F. and Felgner, P.L. (1993) Cationic lipid mediated delivery of polynucleotides. Methods: A Companion to Methods in Enzymology 5, 67-75. 13. Lee, J.T. and Jaenisch, R. (1996) A method for high efficiency YAC lipofection into murine embryonic stem cells. Nucl. Acids Res. 24, 5054-5. 14. Lamb, B.T. and Gearhart, J.D. (1995) YAC transgenics and the study of genetics and human disease. Curr. Opin. Genet. Dev. 5, 342-8. 15. Wilson, T., Papahadjopoulos, D. and Taber, R. (1979) The introduction of poliovirus RNA into cells via lipid vesicles (liposomes). Cell 17, 77-84. 16. Malone, R.W., Felgner, P.L. and Verma, I.M. (1989) Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA 86, 6077-81. 17. 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, 4069-78. 18. 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, 327-41. 19. 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, 2929-31. TEL:03-3669-7980, prometec@jp.promega.com 11