pSINsi-hU6 DNA
Contents
1. DNA RetroNectin PrimeGel TIafd4R4 Tel 077 565 6999 Fax 077 565 6995 http www takara2. RetroNectin VI 3 3 1 1 000 1 8 ug ml 3 4 2 125 250 ul cm2 CO02 3 4 6 http wwwr takara bio coJp 10 3660 3662 VI 4 DSINsi G418 G418 24 3 4 G418
3. 5 10 20 1 2 37C 5 CO2 7 11 7 11 3 ml 10 FBS DMEM 4 ml 2 24 10 FBS DMEM 4 ml http wwwr takara bio coJp 8 3660 3662 3 48 0 45 um 80
4. 5 LTR mRNA pol ll RNA SV40 RNA Dicer siRNA RNA 6 U6 5 RNA 3 UUUU Dier siRNA UUUU pSINsi 3 PSINsi hH1 DNA 3660 human H1 promoter Accession S68670 pSINsi hU6 DNA 3661 human U6 promoter Accession X07425 pSINsi mU6 DNA 3662 mouse U6 promoter Accession X06980
5. pSINsi RNA polymerase ll pol Il RNA pol RNA DNA siRNA Zn HCMV IE promoter MLVR SD Hind ll Nol w Amp coR promoter Hind 1 human H1 human U6 DSINsi hH1 DNA 6 355 bp pSINsi hU6 DNA 6 522 bp pSINsi mU6 DNA 6 573 bp BamHl mouse U6 Ori stuffer EcoR Aco MLV U5 SV40 MLVR r romoter deleted U3 Neo P 3 LTR 1 259 dl FcoR Ca 9g7 dam methylase 1 259 Cg1
6. BLAST http wwwr takara bio coJp 5 3660 3662 IV pSINsi vector RNA DNA IV 1 IV 2 IV 3 IV 4 IV 5 10 X 100 mM Tris HCI pH 8 0 500 mM NaCl gz7 HI 1010A Clal 1034A 1 Awr 1022A PrimeGel Agarose LE 1 20K GAT 5801A DNA NucleoSpin Gel and PCR Clean up 740609 10 50 250 DNA Ligation Kit Ver 1 6021 DNA Ligation Kit Ver 2 1 6022 DNA Ligation Kit lt Mighty Mix gt 6023 E co7JM109 Competent Cells 9052 co7 DH5 a Competent Cells 9057 E coli HST08 Premium Competent Cells 9128 dam LB Amp
7. RNAi siRNA siRNA Design Support System pSINsi siRNA siRNA EN DNA FAX DNA Ce gamH I Cg Top strand Bottom strand BLAST
8. 100 ug ml LB Amp 100 ug ml DNA 1 DNA Top strand Bottom strand 20 pmol ul 1 X 2 95 5 30 25 DSINsi vector 2 ug 4 uD BamH 10 units cal 10 units 10 x K Buffer 2ul up to 20 ul 20 ul 30 1 920 bp hNU6 mU6 vector 5 6 kb hH1 vector 5 4 kb 10 20 ul TE 1 1 ul DNA NucleoSpin Gel and PCR Clean up PrimeGel Agarose LE 1 20K GAT
9. IV 7 DNA DNA pSINsi high copy 25 40 ml 100 ug NucleoBond Xtra Midi 740410 10 50 100 1 mg ml http wwwr takara bio coJp 7 3660 3662 V V 1 Retrovirus Packaging Kit Eco Ampho 6160 6161 G3T hi 6163 293 293T 5 ml DMEM 4 5 g L Glucose with L Glutamine Fetal Bovine Selum FBS Penicillin Streptomycin Mixture Hexadimethrine bromi
10. IV 5 1 DNA IV 4 1 ul DNA 3 pmol TE 5 ul http wwwr takara bio coJp 6 3660 3662 IV 5 2 DNA Ligation Kit Ver 1 1 DNA 5 ul A 20 ul ES B 5 ul 2 16 30 DNA Ligation Kit Ver 2 1 1 DNA 5 ul 5 2 16 30 DNA Ligation Kit lt Mighty Mix gt 1 DNA 5 ul Ligation Mix 5 2 16 30 IV 5 3 1 10 ul 100 ul 2 LB Amp 37 16 IV 6 1 IV 5 3 2 5 mlLB Amp
11. RetroNectin G418 G418 2 G418 G418 VII Retrovirus Packaging Kit Eco Ampho 6160 6161 G3T hi 6163 Retrovirus Constructive System Eco Ampho 6164 6165 RetroNectin Recombinant Human Fibronectin Fragment T100A B RetroNectin Dish RetroNectin Pre coated Dish 35 mm o T110A Retrovirus Titer Set for Real Time PCR 6166 PrimeGel Agarose LE 1 20K GAT 5801A NucleoSpin Gel and PCR Clean up 740609 10 50 250 NucleoBond Xtra Midi 740410 10 50 100 DNA Ligation Kit Ver 1 6021 DNA Ligation Kit Ver 2 1 6022 DNA Ligation Kit lt Mighty Mix gt 6023 E coli JM109 Competent Cells 9052 E coli DH5 a Competent Cells 9057 E coli HST08 Premium Competent Cells 9128
12. 000 gt 3662 IdfaR4 DSINsi hH1 DNA 3660 DSINsi hU6 DNA 3661 DSINsi mU6 DNA 3662 vV201510Da VI VII VIII 1 RNA DNA 2 pSINsi RNA DNA IV 1 IV 2 IV 3 DNA IV 4 IV 5 IV 5 1 DNA IV 5 2 IV 5 3 IV 6 IV 7 DNA Retrovirus Packaging Kit Eco Ampho V 1 V 2 V 3 VI 1 VI 2 VI 3 VI 2 RetroNectin VI 2 1 VI 2 2 VI 2 3 VI 3 VI 3 1 VI 3 2
13. 293T 10 106 cfu ml V 3 NIH 3T3 6 5 x 104cells well 1 1 80 37 2 10 DMEM 10 102 103 104 105 200 ul 3 NIH 3T3 8 9 ug ml 10 900 4 10 102 103 104 105 100 102 103 104 105 106 8 ug ml 5 4 6 10 1 ml 2 6 24 G418 500 ug ml
14. 6166 RT PCR NNA http wwwr takara bio coJp 9 3660 3662 VI VI 1 VVI 2 VI 3 CO2 VI 2 RetroNectin VI 2 1 RetroNectin Recombinant Human Fibronectin Fragment T100AB 2 BSA PBS PBS RetroNectin Dish RetroNectin Pre coated Dish 35 mm o T110A VI 2 2 RetroNectin Dish RetroNectin 2 BSA PBS PBS Hank s RetroNectin VI 2 3 1 RetroNectin 125 250 ul cm2 32
15. 10 2 ml 7 3 4 G418 9 12 10 14 PBS 0 2 1 1 ml PBS PBS 1 0 5 ml 0 2 10 10 100 cfu m Retrovirus Titer Set for Real Time PCR
16. 37 16 2 500 ng BamHI B n 1 2 340 bp 80 90 DNA 3 pSINsi Primer R1 pSINsi Primer R2 GGATCC ATCG ATTCCGGAAC CCTAGG TAGCTAAGGCCTTG GATCTCGAAA CGCATGCATCTCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC CTAGAGCTTT GCGTACGTAG AGTTAATCAGTCGTTGGTCC ACACCTTTCA GGGGTCCGAG GGGTCGTCCG dd G TCGTTGGTCC ACACCTTTC pSINsi Primer R1 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC TCTTCATACGTTTCGTACGT AGAGTTAATC AGTCGTTGGT ATCAGGGCGG GGATTGAGGC GGGTAGGGCG dd GTTTCGTACGT AGAGTTAAT pSINsi Primer R2 DNA pSINsi Primer R1 pSINsi Primer R2
17. 20 ug 500 ng ub 10mM Tris HCIL pH8 0 1 mM EDTA http www takara bio coJp 4 3660 3662 Il 20 2 lll 1 RNA DNA RNA Ligation Ligation DNA pSINsi PamH Cg 1 DNA Top strand Bottom strand 2 N pollll G A G A G A CTGTGAAGCCACAGATGGG Boden eta GTGTGCTGTC
18. C http www takara bio coJp 3 3660 3662 siRNA Retrovirus Packaging Kit Eco Ampho 6160 6161 293 293T siRNA siRNA siRNA pSINsi 3 LTR U3
19. VH 3 3 VI 4 http www takara bio co jp 2 3660 3662 1 RNA RNA interference RNAi RNAi RNA mRNA 21 23 RNA short interfering RNA siRNA RNAi RNAi siRNA siRNA siRNA RNAi RNAi
20. 37 CO 2 3 5 RetroNectin RetroNectin 2 0 2 1X10 cells ml 3 RetroNecttin PBS 1 4 0 5 2 5 X104 cells cm2 VI 3 VI 3 1 8 mg ml VI 3 2 0 5 2 5 104 cells cm lt
21. BamHT 1010A clal 1034A BIn TAvr ll 1022A pSINsi DKIDNA Set 3663 DSINsi DK II DNA Set 3664 VII 1 Boden er al 2004 Nucleic Acids Res 32 1154 1158 2 Miyagishi er g 2004 Gene Med 6 715 723 3 Lee et g 2002 Nat Biotech 20 500 505 4 Paddison et g 2002 Genes and Dev 16 948 958 5 Paul er g 2002 Nat Biotech 20 505 508 6 Sui er g 2002 Proc Natl 4cgg 5 USA 99 5515 5520 7 Kawasaki H et al 2003 Nucleic Acids Res 31 700 707 http wwwr takara bio coJp 11 3660 3662 IX
22. C Miyagishi et g Lee er g 3 Paddison er g Paul er a 5 Sui er ag 6 TTTTTT T 4 pollll siRNA T 4 DNA Top strand T 4 HI J target seduence sense Hairpin loop target seduence antisense Terminator Cal Top strand 5 GATCC GA NNNNNNNNNNNNNNNNNNN CTGTGAAGCCACAGATGGG NNNNNNNNNNNNNNNNNNN C T TTTTTT AT Bottom strand 3 G C T NNNNNNNNNNNNNNNNNNN GACACTTCGGTGTCTACCC NNNNNNNNNNNNNNNNNNN G A AAAAAA TAGC G A G A 2 RNAi
23. bio coJp v201510Da
24. de Aldrich 8 mg ml NIH 3T3 0 45 um V 2 G3T hi 293 293T 6 cm 3 x 106 cells 1 1 Retrovirus Packaging Kit pGP pE eco ampho 2 3 10 FBS DMEM 1 000 1 37 4 70 80 3 ml 5 DNA 5 ml PSINsi retrovirus vector 1 ug ul 10 ul pGP Vector 5 ul DPE eco ampho Vector 5 ul 2 M CaCl2 62 wl 418 6 500 ul
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