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TArget Clone TM

1. 30 2 4C 12 18 25 25C DNA PCR PCR TA PCR UV PCR lacZ IPTG X gal PCR DNA
2. 10 30 2 10 3 Step1 Step2 Step3 pTA2 Vector 2x Ligation Buffer T4 DNA Ligase pTA2 Vector 2x Ligation Buffer pTA2 Vector dT 10 OU 2x Ligation Buffer 50 pTA2 Vector 50ng PR Xul T4 DNA Ligase 10 gl PCR DNA
3. PCR 3 dA overhangs PCR 72 C 5 10 3 dA overhangs PCR 20 PCR DNA PCR DNA PCR PCR PCR 3 dA PCR 2 3
4. pTA2 Vector PCR 1 3 pTA2 Vector 50ng l 3 0kb X PCR X 50Y Z Ykb PCR Z ng 1 PCR 20ng l PCR 0 5kb pTA2 Vector PCR 1 3 50 x 0 5 20 1 25 1 PCR 15 25 C 30 DNA 2kb 5 1218 4 4 Competent high JM109 Code No DNA 900 Competent h
5. 3 dA overhangs DNA 3 dA 0 Taq Tth PCR 3 dA overhangs PCR 72 C 5 10 3 dA overhangs PCR PCR 20 10x A attachment Mix dA KOD PCR PCR PCR dA PCR dA p 4 PCR DNA fragment MagExtractor PCR amp Gel Clean up dT pTA2 Vector
6. 3 dA 3 dT T PCR dA PCR TA TA PCR TA TA TArget Clone
7. 1 2 1 5 PCR DNA pTA2 PCR 1 3 PCR PCR DNA fragment MagExtractor PCR amp Gel Clean up PCR UV PCR UV LB IPTG X gal IPTG X gal PCR 3 PCR PCR
8. IPTG X gal LB 100mM IPTG 4 X gal 20 2 Code No 10 x A attachment mix 25ul 25 TAK 301 T4 DNA Ligase 400Ux1 LGA 111 rTag DNA Polymerase lt Mg gt 250U x 1 TAP 201 rTag DNA Polymerase lt Mg gt 250U x 1 TAP 211 rTth DNA Polymerase 250U x 1 TTH 301 KOD FX 200Ux1 KFX 101 KOD Plus 200Ux1 KOD 201 KOD Plus Ver 2 200Ux1 KOD 211 KOD Dash 250Ux1 LDP 101 Blend Taq 250Ux1 BTQ 101 Blend Taq Plus 250Ux1 BTQ 201 MagExtractor Plasmid Re Plasmid DNA gt a MagExtractor PCR amp Gel Clean up hk RN gt i EO Magical Trapper lt gt 1 MGS 101 Competent high DH5 gw lt DH5 y SOC gt MEN Insert Check Ready 1mlx5 lt coh PCR Premix HPCR 530 8230 2 8 TEL 06 6348
9. PCR PCR DNA fragment MagExtractor PCR amp Gel Clean up DNA 50bp PCR 6 1 LB 10g bacto tryptone 5g bacto yeast extract 10g NaCI 1L NaOH pH7 2 LB LB 15g 50 100 9 ml 100mM IPTG 0 24g IPTG 10ml 20 C 4 X gal 0 4g X gal 5 bromo 4 chloro 3 indolyl 8 D galactoside N N dimethyl formamide 10ml 20 LB
10. 1hr Insert Check Ready PCR 1 gt 1 5hr Pick up a 1hr 4 1 pTA2 Vector Map 1 nri lacz Amp KpnI pTA2 2981 bp rT MCS SacI lac promoter pUC ori 2 4pg 1 Hinc II 7g II AccI Bspl1061 gssH TI T7 Promoter Kpnl Eco0109I XhoI Sall Clal Hind I EcoRYV 1 1 KS primer binding site M13 20 primer binding sit T7 primer binding site 5 TTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATAT 3 AACATTTTGCTGCCGGTCACTCGCGCGCATTATGCTGAGTGATATCCCGCTTAACCCATGGCCCGGGGGGGAGCTCCAGCTGCCATAGCTATTCGAACTATA EcoRI Pstl SmalBamHI Spel Xbal Notl BsIXISacI Sgc1 EcoR I a oa dE OT d insert GTATTGGGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCC GCTTAAGGGTTATG TCATAACCCTTAAGGACGTCG6GCCCCCTAGGTGATCAAGATCTCGCCGGCG6TGGCGCCACCTCGAGG SK primer binding site ssH II AGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCC 3 fl origin 21 327 TCGAAAACAAGGGAAATCACTCCCAATTAACGCGCGAACCGCATTAGTACCAGTATCGACAAAGG 5 galactosidase fragment 460 836 3 ns i multiple cloning site 653 780 T3 primer binding site M13 Reverse primer binding site lac Promoter 837 958
11. 5 12kb KOD DNA w DNA PCR TA PCR Tag DNA 3 dA KOD PCR TA A attachment Mix TA KOD TA TArget Clone
12. 24 C 5 E coli DH5 1 x 10 cfu 9 SN ww ww rTaq TAK 101 KOD TAK 201 5 PCR DNA PCR PCR Premix Insert Check Ready Code No PIK 101 2 4 Insert Check Ready Insert Check Ready Code No PIK 101 gt Step1 Insert Check Ready PCR 50 gk Step2 lt 37 C
13. Stcp3 PCR Step4 PCR Step5 PCR Loading Dye DNA DNA DNA DNA DNA MagExtractor Plasmid Overnight Pick up Mini Prep 1 5hr RNase 0 5 gt 1 5hr
14. GTGCAAAAAA AGTTGGCCGC CTTACTGTCA AACCAAGTCA C6GCGTCAAT CTCATCATTG GCTGTTGAGA CAGCATCTTT CAAAATGCCG CATACTCTTG TCATGAGCGG GTTCCGCGCA T6AGGCACCT GACTCCCCGT CCCAGTGCTG ATCAGCAATA CAACTTTATG GTAAGTAGTT AGGCATCGTG GTTCCCAACG GCGGTTAGCT AGTGTTATCA T6CCATCCGT TTCTGAGAAT ACGGGATAAT GAAAACGTTC TCCAGTTCGA TACTTTCACC CAAAAAAGGG CTTTTTCAAT ATACATATTT CATTTCCCCG dT ATCTCAGCGA CGTGTAGATA CAATGATACC AACCAGCCAG C6CCTCCATG CGCCAGTTAA GTGTCACGCT ATCAAGGCGA CCTTCGGTCG CTCATGGTTA AAGATGCTTT AGTGTATGCG ACCGCGCCAC TTCGGGGCGA TGTAACCCAC AGCGTTTCTG AATAAGGGCG ATTATTGAAG GAATGTATTT AAAAGTGCCA TCTGTCTATT ACTACGATAC GCGAGACCCA CCGGAAGGGC CAGTCTATTA TAGTTTGCGG CGITCGTTTGG GTTACATGAT TCCGATCGTT T6GCAGCACT TCTGTGACTG GCGACCGAGT ATAGCAGAAC AAACTCTCAA TCGTGCACCG GGTGAGCAAA ACACGGAAAT CATTTATCAG AGAAAAATAA TCGTTCATCC GGGAGGGCTT CGCTCACCGG CGAGCGCAGA ATTGTTGCCG AACGTTGTTG TATGGCTTCA CCCCCATGTT GTCAGAAGTA GCATAATTCT GTGAGTACTC TGCTCTTGCG TTTAAAAGTG GGATCTTACC AACTGATCTT AACAGGAAGG GTTGAATACT GGTITATTGTG ACAAATAGGG 4 Acc65I 1 653 Accl 1 674 AH 1 1173 A gl 1 2061 A w44 2 1487 2733 Alwl 10 739 140 1740 1814 1826 1911 1924 2388 2691 2709 A wNI 1 1584 Apal 659 Apol 3 417 428 701 Aval 2 668 733 Avall 2 2204 2426 BamHl 1 739 Banl
15. dA p 4 74 ge Clone Pus PCR DNA fragment MagExtractor PCR amp Gel Cleanup Code No NPK 601 200 5 PCR 2 3 dA TArget Clone Pus dA 3 P 5 dA Step1 Step2 Step3 Step4 KOD PCR 9 PCR PCR 1x Taq 1 5mM MgCb 0 2mM dNTPs 10x A attachment Mix 1 60 10 30
16. g I 50ul 2x Ligation Buffer 10 T4 DNA Ligase 10u 10x A attachment Mix TArget Clone Plus TAK 201 pTA2 Vector 2x Ligation Buffer pTA2 Vector dT 10 T4 DNA Ligase 10x A attachment Mix 30 15 gt LB 100mg ml 100mM IPTG 4 X gal 3 1 PCR Step1 7g9 DNA 7th DNA KOD DNA Step2 SteD3 DNA
17. pUC origin 1178 1845 T3 Promoter B gal fragment ampicillin resistance bla ORF 1996 2853 3 pTA2 2981 bp CTGACGCGCC CTGTAGCGGC 5 1 10 1 151 201 251 301 351 401 451 501 55 1 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251 1301 1351 1401 1451 1501 1551 1601 1651 1701 1751 1801 1851 1901 1951 CGCAGCGTGA TTTCTTCCCT TAAATCGGGG GACCCCAAAA CTGATAGACG GTGGACTCTT TCTTTTGATT TGAGCTGATT TTACAATTTC CGGTGCGGGC GCAAGGCGAT TAAAACGACG TGGGTACCGG GAATTCCCAA TCTAGAGCGG GAGGGTTAAT TGAAATTGTT AAAGTGTAAA CGTTGCGCTC CATTAATGAA CTCTTCCGCT GGCGAGCGGT TCAGGGGATA CAGGAACCGT CCCCTGACGA CCGACAGGAC GCGCTCTCCT TCCCTTCGGG AGTTCGGTGT CGTTCAGCCC ACCCGGTAAG ATTAGCAGAG GCCTAACTAC TGAAGCCAGT CAAACCACCG GCGCAGAAAA CTGACGCTCA TTATCAAAAA TAAATCAATC CCGCTACACT TCCTTTCTGG GCTCCCTTTA AACTTGATTA GTTTTTCGCC GTTCCAAACT TATAAGGGAT TAACAAAAAT CATTCGCCAT CTCTTCGCTA TAAGTTGGGT GCCAGTGAGC GCCCCCCCTC GCATTAAGCG TGCCAGCGCC CCACGTTCGC GGGTTCCGAT GGGTGATGGT CTTTGACGTT GGAACAACAC TTTGCCGATT TTAACGCGAA TCAGGCTGCG TTACGCCAGC AACGCCAGGG GCGCGTAATA GAGGTCGACG TAC GTATTGGGAATTCCTGC CCGCCACCGC TGCGCGCTTG ATCCGCTCAC GCCTGGGGTG ACTGCCCGCT TCGGCCAACG TCCTCGCTCA ATCAGCTCAC ACGCAGGAAA AAAAAGGCCG GCATCACAAA TATAAAGATA GTTCCGACCC AAGCGTGGCG AGGTCGTTCG GACCGCTGCG ACACGACTTA CGAGGTATGT GGCTACACTA TACCTTCGGA CTGGTAGCGG AAAGGATCTG GTGGAACGAA GGATCTTCAC
18. 3786 FAX 06 6348 3833 E mail order_lifescience toyobo jp 104 8345 17 10 TEL 03 6887 8819 FAX 03 6887 8951 E mail order_lifescience toyobo jp TEL 06 6348 3888 FAX 06 6348 3833 9 00 12 00 13 00 17 00 E mail tech_osaka toyobo jp URL http www toyobo co jp bio
19. 4 193 653 917 2014 Banll 3 159 659 775 Pc VI 2 1382 2909 Bfal 6 81 146 752 1668 1921 2256 Ball 2 466 2180 Bpml 2 778 2151 BsaAl 1 232 saHI 1 2602 Bsal 1 2133 Bsadl 6 575 733 734 767 912 1333 saWI 3 1379 1526 2357 BseMII 4 1448 1857 2023 2563 BsiE 6 497 758 1086 1510 2433 2582 BsiHKAI 4 715 1487 2648 2733 Bsll 8 9 335 664 1015 1189 1207 1373 1652 BsmAl 2 2133 2898 Bsp1201 1 659 Bsp12861 6 159 659 775 1487 2648 2733 gspHI 2 1893 2901 spLU111 1 1173 ps BI 5 88 755 863 1104 2905 gs DI 2 2120 2302 ssHII 2 619 812 pssSI 2 1346 2730 gs F5I 4 542 2046 2227 2514 BstXxl 1 764 C 101 2 129 2146 C al 1 683 Csp8l 2 654 2545 Ddel Dral Dralll Dral Dsal Eael Earl Ecl13611 Eco521 co57 coO1091I coRI coRII coRV Faul Fokl Fspl Haell Hgal Hincll Hindlll Hinfl Hphl Kpnl Maell Mboll Msll MspAll Mval Nael Ncil NgoMIV Nlalll Noil sl Ple Psp14061 Pstl Pvaul Pvull Rsal Sacl Sacll Sall oo PR ON CO Co Sh 5 S S 1448 1930 232 275 767 609 511 775 758 1700 659 701 575 695 33 542 477 15 674 689 282 1148 222 2781 653 123 1876 103 2669 765 527 2699 575 129 657 2599 129 828 2473 757 1173 282 2061 2291 727 497 527 6
20. 54 775 767 674 1857 1949 1275 758 1051 2748 721 912 87 2046 2286 1275 304 1544 1917 233 2292 512 2747 2314 167 912 133 1174 2509 304 2664 2433 995 2545 2023 2641 1012 2855 1200 505 2227 1047 1853 632 2061 2144 276 2665 1052 2856 2473 995 1200 734 1894 2902 632 2563 2454 1321 965 2514 1417 2603 1008 2540 288 1823 2832 1513 1321 1552 2385 1073 1334 1022 1073 2766 595 1914 1758 1334 2248 2395 1544 Sapl Sa096 Scal Schl SfaNl Sfcl Smal Smll Spel Sspl Taal Tail Tagl Tatl 7sp45 7 spRI Vspl Xbal Xhol Xholl Xmal Xmnl CO oP ho Co Co CO o gt 1050 240 2187 2544 282 2061 1261 11 2307 133 668 745 440 258 1676 123 1876 199 1273 2544 1008 57 613 1859 943 751 668 739 2691 733 2661 507 659 2204 2426 304 632 2313 2523 638 121 1279 1541 2868 483 678 1989 2504 233 276 2292 2665 669 675 2717 1148 589 2323 960 1069 2008 2113 1002 2237 1814 1825 2708 10 Aatll Bbel Binl Bsgl spMI BstEll Ehel Ms
21. Plus A attachment Mix KOD PCR A attachment Mix 60C 10 PCR Tag DNA PCR TA A attachment 2 DNA A2kb PCR PCR product 100 Method 1 7gg PCR 90 Method 2 KOD PCR Method 3 KOD PCR A attachment KOD TT KOD rtailing by A attachment Mix 60C 10 min Ga dA ca S 1 3 0 KOD KOD Method 7ag PCR dA Cloning efficiency ow Ligation to PTA2 7ag PCR 2 10l pTA2 Vector 50ng AD TArget Clone TAK 101 50l 2x Ligation Buffer 10u T4 DNA Ligase 10 20C 10l pTA2 Vector 50ng
22. TAAAGTATAT GGTGGAGCTC GCGTAATCAT AATTCCACAC CCTAATGAGT TTCCAGTCGG CGCGGGGAGA CTGACTCGCT TCAAAGGCGG GAACATGTGA CGTTGCTGGC AATCGACGCT CCAGGCGTTT T6CCGCTTAG CTTTCTCATA CTCCAAGCTG CCTTATCCGG TCGCCACTGG AGGCGGTGCT GAAGGACAGT AAAAGAGTTG T6GTTTTTTT AAGAAGATCC AACTCACGTT CTAGATCCTT ATGAGTAAAC CGGCGGGTGT CTAGCGCCCG CGGCTTTCCGC TTAGTGCTTT TCACGTAGTG GGAGTCCACG TCAACCCTAT TCGGCCTATT TTTTAACAAA CAACTGTTGG T6GCGAAAGG TTTTCCCAGT CGACTCACTA GTATCGATAA AGCCCGGGGG CAGCTTTTGT GGTCATAGCT AACATACGAG GAGCTAACTG GAAACCTGTC G6CGGTTTGC GCGCTCGGTG TAATACGGTT GCAAAAGGCC GTTTITTCCAT CAAGTCAGAG CCCCCTGGAA CGGATACCTG GCTCACGCTG GGCTGTGTGC TAACTATCGT CAGCAGCCAC ACAGAGTTCT ATTTGGTATG GTAGCTCTTG GTTTGCAAGC TTTGATCTTT AAGGGATTTT TTAAATTAAA TTGGTCTGAC GGTGGTTACG CTCCTTTGGC CGTCAAGCTG ACGGCACCTC GGCCATCGCC TTCTTTAATA CTCGGTCTAT GGTTAAAAAA ATATTAACGC GAAGGGCGAT GGGATGTGCT CACGACGTTG TAGGGCGAAT GCTTGATATC ATCCACTAGT TCCCTTTAGT GTTTCCTGTG CCGGAAGCAT ACATTAATTG GTGCCAGCTG GTATTGGGCG GTTCGGCTGC ATCCACAGAA AGCAAAAGGC AGGCTCCGCC GTGGCGAAAC GCTCCCTCGT TCCGCCTTTG TAGGTATCTG ACGAACCCCC CTTGAGTCCA TGGTAACAGG TGAAGTGGTG TGCGCTCTGC ATCCGGCAAA AGCAGATTAC TCTACGGGGT GGTCATGAGA AATGAAGTTT AGTTACCAAT 2001 2051 2101 2151 2201 2251 2301 2351 2401 2451 2501 2551 2601 2651 2701 2751 2801 2851 2901 2951 GCTTAATCAG ATAGTTGCCGT ACCATCTGGC CTCCAGATTT AGTGGTCCTG GGAAGCTAGA CCATTGCTAC TTCAGCTCCG
23. TOYOBO ldeas amp Chemistry Convenient TA Cloning System for PCR Products TArget Clone Code No TAK 101 TArget Clone Plus Code No TAK 201 TOYOBO CO LTD Life Science Department OSAKA JAPAN A3243K MEO ZR A i ES 2 3 dA ikki 3 009 54 SR RA 4 i CR BTAZ Veclor Ma EN 2 SDEA2 KKKRMMAMe 4 5 5 NG Ne 1 Tag DNA 7th DNA PCR
24. cl Nhel Ppul10 Sbfl SnaBl Swal Acclll Bbsl pu101 BsiwWI BsrGl su36 Fsel Munl Nrul PpuMI SexAl Sphl Tth1111 Aflll Bbvcl Bpu1102 BsmB l Bst11071 Eco47lll Hpal Narl Nsil PshAl Sfil Srfl Va79 11 Agel Bcll BsaBl BsmFI s7 API Eco721 Kasl Ncol Pacl Rsrll Sg Stul Xcml 660 2108 1073 1544 2753 1438 1629 1818 2686 1135 1206 288 595 684 699 2534 1575 1588 2460 2487 1911 1923 Ascl Bglll BseRI Bsml BstBl EcoNl MIiul Ndel Pmel SanDIl SgrAl Styl 5 10 cfu g pBR322 50 100 g ml 50 100 g ml 4C 1 PCR Taqd TtIh
25. igh DH5 w Code No DNA 903 Step1 Competent Cell 100 Step2 10 I 30 Step3 42 30 2 Step4 SOC 900 Il 37 1 Step5 LB IPTG X gal 37 1 20 100 100 go 500bp 1kb 2kb 80 eR DNA 70 5oobp tk vo 60 rTaq 10 400 4 700 1 800 9 2 TAK 101 ee 40 KOD Plus 8 950 4 300 2 230 BS TAK 201 20 10

TArget Clone TM

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