取扱説明書 - 東洋紡ライフサイエンス事業部
Contents
1. jn vitro dam methylase dam Plasmid 4 1 LB IPTG X gal IPTG X gal DH5 amp JM109 7 1 LB 1L 10g bacto tryptone 5g bacto yeast extract 10g NaCI 1L NaOH pH7 2 LB 1L LB 159 50 C 100 g ml LB 1L LB2. PCR Inverse PCR 108 cfu hg pBR322 jn vitro dam methylase dam Plasmid 4 1 4 C 1 PCR PCR
3. 3 SelEligation 10 30 pBR322 pBR322 1pg 100 Il 1x10 cfu ug pBR322 100 42 C 30 2 SOC 900 37 C 1 LB 10 200 LB IPTG gal LB IPTG X gal 37 C 1 16hr
4. Inverse PCR PCR HPLC 5 HPLC 40mer PAGE ET a 1 A 2 Substitution 5 5 3 20bp
5. 6 Plasmid Plasmid Inverse PCR Plasmid Inverse PCR PCR Inverse PCR Plasmid IPTG X gal
6. PCR 10 20 PCR PCR 4 PCR 2nd site mutation PCR KOD Plus 2nd site mutation 2nd site mutation 5 Plasmid Plasmid lacZ o Plasmid pAK119M
7. PCR 2 5 3 20bp 25bp PCR PCR KOD Plus 3 5 Exonuclease Proofreading
8. 3 PCR Self ligation T4 Polynucleotide Kinase Ligation high Ligation highn PCR p7 1 PCR 2ul 7 l Ligation high 5 l T4 Polynucleotide Kinase Tul Total Volume 15 AI1 16 1 4 Competent high DH5 w Code No DNA 903 Competent high JM109 Code No DNA 900 Competent Cell 100
9. lacZ w 6 TAA stop Plasmid X gal TAA stop CCA Pro 3 809 Plasmid 2 pAK119M 4 3 kb ColE ori lacZw ss TAA stop White Colony CCA Pro Blue Colony LacZo gt N MetThrMetlleThrSTOP C AATTTCACACAGGAAACAGCTATGACCATGATTACGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATC TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCATTCGAACGTACGGACGTCCAGCTGAGATCTCCTAG Control Primer 1 5 CCAAGCTTGCATGCCTGCAGGTCGA 3 AATTTCACACAGGAAACAGCTATGACCATGATTACGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATC TTAAAGTGTGT
10. KOD Plus PCR PCR 2nd site mutation 10kbp 3 PCR Selfligatton Kinase Ligase PCR Primer 3 1 2 Inverse PCR CH3NCH3 CH3 CH3 Template CH3 TAL Inverse PCR 5 10 lt 0 5 2hr gt CH3 CH3 Template x Dpn 1 plasmid lt 37 c 1hr gt PCR Self ligation Kinase Ligase 16 1hr gt DL Transform E coli 1 Inverse PCR A Plasmid DNA Inverse PCR
11. 1x10 cfu hg pBR322 100 100 809 5 4 lt 8 Plasmid Plasmid PCR
12. B DNA Zpz1 Plasmid DNA C T4 Polynucleotide Kinase Ligase PCR D PCR 3 20 No 20 1 KOD Plus 1U g 1 25 2 10x Buffer for iPCR 125 3 2mM dNTPs 125 4 Dpn 10U 50 5 T4 Polynucleotide Kinase 5U 1 50 l 6 Ligation high 250 7 Control Plasmid pAK119M 50ng AD 10 8 Control Primer 1 10 pmol AD 10 9 Control Primer 2 10 pmol AD 10 20 5 gt LB 50mg ml 20mg ml 4 X gal 100mM IPTG Plasmid Blue White Competent high DH5 w Code No DNA 903 JM109 Code No DNA 900 4
13. NPK 601 lt DNA fragment gt Magical Trapper lt gt 1 MGS 101 Competent high DH5 gw 100 x10 w MUI x DNA 903 lt Eco DH5 SOC gt Competent high JM109 8 100l x10 DNA 900 lt Eco JM109 SOC gt 13 530 8230 2 8 TEL 06 6348 3786 FAX 06 6348 3833 E mail order_lifescience toyoboJp 141 8633 10 2 TEL 03 6422 4819 FAX 03 6422 4951 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
14. Inverse PCR iPCR Inverse PCR DNA 2 PCR PCR PCR PCR Selfligation 1 Inverse PCR bp 10bp Tag 100bp 20 Mutant Clone Collection Saturation Mutagenesis 2 95
15. 159 50 C 20 gIml 100mM IPTG 10mL 0 24g IPTG 10ml 20 4 X gal 10mL 0 4g X gal 5 bromo 4 chloro 3 indolyl 8 D galactoside N N dimethyl formamide DMF 10ml 20 LB IPTG X gal LB 100mM IPTG 49 X gal 20 2 Code No KOD Plus lt PCR gt 200Ux 1 KOD 201 200Ux 1 x5 KOD 201X5 200Ux 1 x 10 KOD 201X10 p 1 000Ux1 DPN 101 1 000Ux 1 x 5 DPN 101X5 T4 Polynucleotide Kinase 1 500Ux 1 PNK 111 1 500U x 1 x 5 PNK 111X5 Ligation high lt gt 50 LGK 101 MagExtractor Plasmid 500 NPK 301 lt Plasmid DNA gt MagExtractor PCR amp Gel Clean up 2 200
16. 3 35l 10 x Buffer for iPCR 5 2 mM dNTPs 5l 0 Control Primer 1 10 pmol D 1 5l 5 68C 5min Control Primer 2 10 pmol AD 1 5 5 Control Plasmid pAK119M 50ng gu I 1l KOD Plus 1 Total Volume 50w 2 Dpn Plasmid PCR 50 p 1 2 PCR 2 PCR pnl1 37 C 1 PCR Dpn 6 PCR 5
17. Plasmid DNA 50ng Il PCR 35 10 x Buffer for iPCR 5 1 2mM dNTPs 5 1 10 pmol 1 5 2 10 pmol 1 5 Plasmid DNA 50ng 1l KOD Plus 1 Total Volume 50 PCR 94C 2min 98C 10sec 68C Xmin 4C Hold YG 10 2 1 kb 1min kb 5kb Plasmid 5min 2 kb 1 kb 2 5 10 PCR PCR 10 20
18. 25bp Deletion Insertion 5 3 20bp 25bp 3 PCR Inverse PCR PCR iPCR buffer 2 PCR Plasmid
19. 1 Plasmid PCR Plasmid Dpn pn G ATC DNA Plasmid DNA JM109 DH5 w Plasmid dam methylase dam JM110 SCS110 Plasmid Plasmid in vitro dam methylase JM109 dam Plasmid 2 PCR Primer
20. CCTTTGTCGATACTGGTACTAATGCATTCGAACGTACGGACGTCCAGCTGAGATCTCCTAG 3 CCTTTGTCGATACTGGTACTAATGC 5 Control Primer 2 N MetThrMetlleThrProSerLeuHisAlaCysArgSerThrLeuGluAsp AATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGAT TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCGGTTCGAACGTACGGACGTCCAGCTGAGATCTCCTA 3 Plasmid Control Plasmid pAK119M LacZw galactosidase alpha fragment CC T LacZw 6 TAA stop Plasmid X gal T CC 6 TAA stop CCA LacZo X gal 5 1 Inverse PCR PCR 10 pmol
21. TOYOBO ldeas amp Chemistry KOD Plus Mutagenesis Kit Code No SMK 101 TOYOBO CO LTD Life Science Department OSAKA JAPAN AS3634K di i 1 2 Inverse PCR 2 3 ki 3 4 i 4 1 Plasmid kk 4 2 PCR Primer 4 CSIPCR TS i 5 4 PCR 2nd site mutation 5 5 Plasmid ki 6 DN 8 1 Inverse PCR i 8 2 Dpn 1I Plasmid 9 3 PCR OSelf ligation 9 AR 10 5 10 G 11 1 ii 12 1 ki 12 2 ek 13 1 PCR KOD Plus
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