/ N /C / / Flexi Vector N2391, N2401, N2411 MCS Vector N2361, N2371, N2381 Flexi ORF Wizard SV Gel and PCR Clean-Up System A9281 LigaFast Rapid DNA Li

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1 プロメガ株式会社

2 / N /C / / Flexi Vector N2391, N2401, N2411 MCS Vector N2361, N2371, N2381 Flexi ORF Wizard SV Gel and PCR Clean-Up System A9281 LigaFast Rapid DNA Ligation System M / / / / / Reagent PureYield Plasmid Mini A1223 Midi A2492 Maxi kits A2392 ViaFect E4981, E4982 FuGENE HD E2311, E2312 Nano-Glo Lytic Detection System N3030 Nano-Glo Blotting System N2410 Nano-Glo Extracellular Detection System N2420 GloMax

3 1 aflexi ORF Flexi Vector N2391, N2401, N2411 ORF Flexi Flexi ORF Clone 20,000 or ORF bmcs MCS Vector N2361, N2371,N2381 ORF PCR, / PCR, CRISPR / Cas

4 1 AFlexi Flexi ORF Flexi Vector N2391, N2401, N2411 ORF ORF Flexi Flexi ORF Clone 20,000 Flexi VectorN2391, N2401, N2411 Flexi ORF Clone Flexi Cloning SystemC8640, C9320, C8820 Flexi N and/or C Flexi Vector pfc37k CMV-neo Flexi Vector pfn38k CMV-neo Flexi Vector pfn39k sec CMV-neo Flexi Vector POI: protein of interest POI-VSQGSSGGSSG- -GSSGGSSGAIA-POI IL6--GSSGGSSGAIA-POI BMCS MCS MCS Vector N2361, N2371,N2381 ORF PCR, cdna PCR and/or ORF Wizard SV Gel and PCR Clean-Up SystemA9281 MCS VectorN2361, N2371, N2381 ORF LigaFast Rapid DNA Ligation SystemM8221, M8225 4

5 pbit3.1 Vector,,, MCS pbit3.1-n [CMV//Blast] Vector pbit3.1-c [CMV//Blast] Vector pbit3.1-secn [CMV//Blast] Vector POI: protein of interest -GSSG-POI -GSSGGSSG-POI -GSSGGSSGGNS-POI -GSSGGSSGGNSGGGS-POI POI-GSSG- POI-GSSGGSSG- POI-GNSGSSGGSSG- POI-GSGGNSGSSGGSSG- IL6--GSSG-POI IL6--GSSGGSSG-POI IL6--GSSGGSSGGNS-POI IL6--GSSGGSSGGNSGGGS-POI pbit3.1 Vector MCS pbit3.1-n [CMV//Blast] Vector 5...GCCATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGGAGCTCCGGTGGCTCGAGCGGTGGGAATTCTGGT SbfI NheI BglII XbaI HindIII PmeI GGAGGATCCGGTGGAGCTAGCGGAAGATCTGGTTCTAGAGGTCAAGCTTCGGGGGTTTAAACCTGCAGGCAT...3 pbit3.1-c [CMV//Blast] Vector SgfI HindIII BglII NheI 5...GGGGCGATCGCAACAAGCTTCGGGAAGATCTGGAGCTAGCGGCGGGGGATCCGGTGGGAATTCTGGCTCGAGCGG PmeI TGGGAGCTCCGGTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTAAGTTTAAACGGCC...3 pbit3.1-secn [CMV//Blast] Vector IL-6 signal sequence 5...GCCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCTGCTGCC IL-6 signal sequence TTCCCTGCCCCAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGGAGCTCCGGTGGCTCGAGCGGTGGGAATTCTGG SbfI NheI BglII XbaI HindIII PmeI TGGAGGATCCGGTGGAGCTAGCGGAAGATCTGGTTCTAGAGGTCAAGCTTCGGGGGTTTAAACCTGCAGGCAT MA 5

6 2 / PCR, 1 A oligo oligo Forward NNNNNXXXXXXNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNXXXXXXNNNNN Reverse NNNNNXXXXXXNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNXXXXXXNNNNN Linker oligo PCR Wizard SV Gel and PCR Clean-Up System A9281 LigaFast Rapid DNA Ligation SystemM8221, M8225 6

B In-Fusion HD Cloning Kit www.promega.co.

7 B In-Fusion HD Cloning Kit oligo PCR Forward NNNNNNNNNNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNNNNNNNNNN Reverse NNNNNNNNNNNNNNNXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXNNNNNNNNNNNNNNN N 15 Linker oligo PCR ds-oligo Wizard SV Gel and PCR Clean-Up SystemA9281 In-Fusion HD Cloning Kit HP 7

8 3 CRISPR / Cas9 CRISPR/Cas9 crrna RNAcrRNA tracrrna insert site. crrna Stop Codon PAM ATGGCTAGGTACCGTCATCCGCAATGGCTAGGTAAGTCCGCTATGGATAGGTACC GAPDH Donor DNA template 5' Exon Exon Exon 3' UTR 3' ~80nt Single-Stranded Oligodeoxynucleotide 33nt Sequence TGA ~80nt 3' Homology Arm Stop Codon Homology Arm 10 CRISPR/Cas RNA crrna tracrrna 30,000 1 sgrna 3 70,000 1 Cas9 100 µg / 22,000 Donor DNA nt 50, Tel / Fax prometec@jp.promega.com プロメガ株式会社 Tel Fax Tel Fax Web sitewww.promega.jp 販売店

<4D435388EA C E696E6464>

<4D435388EA C E696E6464> MULTI CONNECTION SYSTEM MCS MULTI CONNECTION MICRO 2.mm 0.08 0.mm 2 / AWG 28 20 MINI 3.mm, 3.81mm 0.08 1.mm 2 / AWG 28 14 MCS MICRO CAGE CLAMP PA6.6 2. mm 320 V () 6A MCS MINI 734 2734 CAGE CLAMP CAGE