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Adachi413Supl - BioTechniques
Short Technical Reports Supplementary Material For: Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1, Kei-ichi Shibahara2, Noritaka Adachi1, and Hideki Koyama1 1Yokohama City University, Yokohama and 2National Institute of Genetics, Mishima, Japan Iiizumi et al. Supplemental Fig BioTechniques 41:XXX-XXX (September 2006) XhoI XhoI NdeI att R3 ccd B att R4 pMC1 pDEST R4-R3 4.1 kb Invitrogen Cat No. :12537-023 r Amp r Amp NdeI digestion Subcloning DT-A PGK-pA pMC1 DT-A 4.33 kb KURABO Cat No. : MK-1003 XhoI digestion AatII att R4 ccd B att R3 PGK-pA DT-A MLS fragment 1 pMC1 r 20 30 40 50 ������������������������������������������������������ 3' 5' 3' ������������������������������������������������������ 5' pDEST R4-R3 DT-A Amp 10 PmeI 5.4 kb AscI I-SceI SwaI PacI (NdeI/XhoI sites are deleted) Aat II digestion Insertion PacI SwaI I -SceI att R4 ccd B att R3 PGK-pA DT-A pMC1 AscI PmeI pDEST DTA-MLS r Amp 5.4 kb (AatII site is deleted) Supplementary Figure S1. pDEST DTA-MLS. DT-A, a gene that codes for a diphtheria toxin A fragment; MLS, multiple-linearization site. Vol. 41 ı No. 3 ı 2006 www.biotechniques.com ı BioTechniques ı 1 Short Technical Reports A A att B1 primer att B2 primer at t B HindIII BamHI BamHI HindIII Hyg r PolyA Hyg r PolyA pPGK pPGK att L1 NotI His r NotI Hyg r PolyA pPGK att L1 yA Km Hyg r pPGK r att L2 pβActin PolyA att L1 His r pβActin NotI His r PolyA pβActin Puro r pPGK Sal I PolyA Subcloning NotI NotI pPGK pBS lox-His 6.2 kb Puro r NotI pPGK PolyA p pBS lox-Puro 4.2 kb NotI digestion Subcloning into pENTR loxP NotI digestion Subcloning into pENTR loxP EcoRV pBS246 pPGK Puro 2.5 kb 4.9 kb EcoRV digestion SalI digestion Subcloning pβActin D BamHI D EcoRV att L1 att L2 PolyA PolyA r Km pβActin His r att L2 PolyA PolyA pPGK att L1 pENTR lox-His 6.6 kb r Km NotI digestion Subcloning into pENTR loxP Puro r att L1 att L2 PolyA pENTR lox-Puro 4.6 kb Puro r pPGK pBS246 2.5 kb PolyA pPGK Puro 4.9 kb EcoRV digestion SalI digestion Subcloning NotI NotI ctin NotI pPGK Puro r PolyA pBS lox-Puro 4.2 kb NotI digestion Subcloning into pENTR loxP digestion oning into pENTR loxP att L2 att L1 s r pPGK Puro r PolyA att L2 pENTR lox-Puro 4.6 kb Km Supplementary Figure S2. Entry clones for positive selectable markers. (A) pENTR loxP; (B) pENTR lox-Hyg; (C) pENTR lox-His; and (D) pENTR loxPuro. Hygr, hygromycin-resistance gene; Hisr, histidinol-resistance gene; Puror, puromycin-resistance gene. 2 ı BioTechniques ı www.biotechniques.com pPGK pEN r Km Sal I SalI pβActin pβActin His 6.4 kb His r pENTR lox-His 6.6 kb r Km bcloning olyA olyA BamHI SalI pβActin pBS246His 6.4 kb kb 2.5 NotI pBS lox-His 6.2 kb pENTR lox-Hyg 4.9 kb pENTR lox-Hyg r 4.9 kb Km His r D att L2 pENTR loxP att L1 2.9 kb att L2 PolyA Hyg r pPGK His r PolyA r BamHI digestion EcoRV digestion NotI NotI digestion Subcloning into pENTR loxP NotI NotI NotI digestion Subcloning into pENTR loxP BP recombination pPGK PolyA Subcloning pBS lox-Hyg 4.6 kb pBS lox-Hyg 4.6 kb att L1 Hyg r PolyA Km EcoRV pβActin BamHI digestion NotI NotI att L2 pENTR loxP 2.9 kb BamHI BamHI BamHI pBS246 pβActin His 2.5 kb 6.4 kb Subcloning Subcloning att L1 att L2 D Iiizumi et al. Supplemental Figure 2 NotI att P2 C C PolyA pPGK-Hyg pBS246 4.9 kb 2.5 kb HindIII, BamHI digestion HindIII, BamHI digestion r BamHI BamHI BamHI ccd B NotI NotI NotI BP recombination NotI pDONR 221 4.8 kb pENTR loxP 2.9 kb Invitrogen r No. : 12537-023 Cat. Km Km C HindIII att B2 att P1 att P2 pDONR 221 4.8 kb Invitrogen Cat. No. : 12537-023 pBS246 pPGK-Hyg 2.5 kb 4.9 kb pBS246 2.5 kb ccd B att P1 r B PCR pBS246 att B1 2.5 kbatt B2 Gibco BP recombination Cat.No. : 10348-019 att B1 Km HindIII BamHI B2 B1 B1 B2 t at PCR pBS246 2.5 kb Gibco Cat.No. : 10348-019 B att B2 primer at t att B1 primer t at A Iiizumi et al.Figure Supp Iiizumi et al. Supplemental Vol. 41 ı No. 3 ı 2006
