Ligation high Ver.2
Contents
1. 30 SS 6 APPENDIX 1 E coli Alkaline Phosphatse 0 5 2 5 g 80 4 10 x Buffer 10 E coli Alkaline Phosphatase 0 1 1U gl 10 Total 100 5 20pmoles 5 2U 10 3 60 C 60min 5 37 C 62. PCR DNA DNA DNA TA APPEDIX PCR TA 2 16 2 DNA DNA
3. X PCR 0 5 1 Ligation high Ver 2 7 5 15 5 p6 gt 4 1 pUC19 Hindlll 50ng 25fmol 4 DNA Hindll 564bp DNA lt 7 5 ux gt Ligation high Ver 2 lt 7 5 gt 16 C 30 10 uu E coli DH5 w lt 100 gt LB Amp X Gal IPTG Vector Insert 1 3 0 1 1 6 AT So2 Vector Insert 3 1 5 100 2 pUC19 cll 50ng 25fmol PCR KOD Plus 0
4. Ligation high Ligation high Ver 2 Ligation high 2 2 Ligation high Ligation high Ver 2 Ligation high Ver 2 DNA Ligation 1 0 5 1 0 5 1 1 6 6 6 pfuwwg ZAPII 5 6x 10 6 3x10 5 5x 10 5 1 10 1710 DNA DNA PCR
5. 16 C 5 109 1 Ligation high Ver 2 DNA Ligation 1 1 or 1 0 5 16 C 5 30 1 1 or 1 0 5 16 30 1 1 or 1 0 5 16 C 30 T 1 1 16 30 PCR T 1 1 16C 30 PCR 1 1 or 1 0 5 16 30 1 lt 2 0 5 1
6. 2 8 TEL 06 6348 3786 FAX 06 6348 3833 E mail order lifescience toyoboJp 104 8345 17 10 TEL 03 6887 8819 FAX 03 6887 8951 E mail order lifescience toyoboJp TEL 06 6348 3888 FAX 06 6348 3833 9 00 12 00 13 00 17 00 E mail tech_osaka toyoboJp URL http www toyobo cojp bio
7. S 8 3 DNA 1 DNA DNA 7 5l Ligation high Ver 2 3 75 7 5 1 16C 30 100 uu 10 gg I 4 TE Buffer 10mM Tris HCIlpH8 0 1mM EDTA DNA 2 TA L 3 2 2 5 4 4C 25 C
8. lt 100 gt LB Amp X Gal IPTG PCR 4 DNA PCR 120 100 80 60 40 0 2 1 0 5 PCR l lt 15 gt 7 TA 0 5 100 6 EcoRI 4 ZAP II 500ng 2 8kb pRheo EcoRI test insert 225ng 5u1 gt 5 uu 1 gt 2 5 gl1 gt 16 C 30min 1 5 GIGAPACKYIII Stratagene coli XL 1 Blue MRF
9. 14 DNA Ligase DNA 1 Ligation high Ligation high Ver 2 Ligation high T4 DNA Ligase Ligation high TA 120 100 ES 80 60 I HT 40 0 a a AT ey WS 1 TA Ligation high Ver 2 100
10. 10mer DNA lt 7 5 gt Ligation high Ver 2 lt 7 5glor 3 75 gt 16 C 30 10 ux E coli DH5 LB Amp X Gal IPTG Vector Linker 1 10 Ligation high Ver 2 1 2 DNA Ligation high Ver 2 1 0 5 120 120 100 100 go 80 1 60 60 FH 40 40 20 20 0 1 1 0 _ 1 1 1 OO Q Vector Insert Vector Insert DNA Ligation high Ver 2 1 1 DNA Ligation high Ver 2 1 0 5 5 DNA Ligation high Ver 2 1 0 5 Vector Insert 1 10 100 4 TA T
11. 37 1h T4 Polynucleotide Kinase 10 2 Code No E co Alkaline Phosphatase 100Ux1 BAP 111 T4 Polynucleotide Kinase 1 500Ux1 PNK 111 rATP 50 moles 0 5ml ATP 111 MagExtractor Plasmid 9 0 500 NPK 301 lt Plasmid DNA gt MagExtractor PCR amp Gel Clean up 9 MR P 200 NPK 601 lt DNA fragment gt Magical Trapper lt gt 1 MGS 101 Competent high DH5 w ee 100lx10 DNA 903 lt Ecoli DH5 w SOC gt Competent high JM109 0 9 a 100lx10 DNA 900 lt EcoliJM109 SOC gt KOD Plus lt PCR gt 200Ux1 KOD 201 KOD Plus Ver 2 lt PCR gt 200Ux1 KOD 211 KOD FX PCR gt 200Ux1 KFX 101 530 8230
12. 50ng 25fmol PCR Taq DNA polymerase 0 5kb DNA lt 7 5 ux gt Ligation high Ver 2 lt 7 5l gt 16 C 30 10 u E co7 DH5 w lt 100 LB Amp X Gal IPTG Vector Insert 1 3 60 LL 1 0 5 1 1 1 3 1 5 Vector Insert 6 TA 1 5 100 5 TA PCR gt T 50ng 25fmol PCR Taq DNA polymerase 0 5kb DNA lt 7 5 gt Ligation high Ver 2 lt 7 5 gt 16 30 10 ux E coli DH5 w
13. 20C Ready to use Ligation high 1 DNA 2 Ligation high Ver 2 750 x 1 1 7 5 100 20C 30 0 5 2 TA 0 100 120 0 So
14. 0min MagExtractorTM PCR amp Gel Clean up Code No NPK 601 T4 polynucleotide Kinase rATP 1 50pmole I M Primer 2 Primer 50pmole 1 50 M 14 10 x Protruding End Kinase Buffer 10mM rATP lt Code No ATP 111 gt T4 Polynucleotide Kinase 5 20U D Total 20 37 1h 95C 5min T4 Polynucleotide Kinase 1 50pl Q 10pmole I 10 M primer 3 PCR IN _ T4 Polynucleotide Kinase 10 PCR PCR T4 polynucleotide Kinase rATP 1 PCR PCR 2 PCR 1 9 35 pb 10 x Blunt End Kinase Buffer 10mM rATP lt Code No ATP 111 gt T4 Polynucleotide Kinase 5 20U gl Total 50
15. 14 03 ldeas amp Chemistry DNA Ligation Kit Ligation high Ver 2 Code No LGK 201 TOYOBO CO LTD Life Science Department OSAKA JAPAN A3706K 2 NZ i NS NA Na Gt RNN 2 kk SU A i A 5 TA PCH gt 6 NN 5 i Bl ABB NILESERKKKM AI 2 5 2 DU PCR EE 1 DNA
16. 5kb ADNA DNA lt 7 5 gt Ligation high Ver 2 lt 7 5l or 3 75 gt 16 30 10 E co7 DH5 gw LB Amp X Gal IPTG Vector Insert 1 3 KOD Plus DNA 120 120 100 100 80 80 60 60 ee 40 40 20 20 0 1 1 0 6 5 9 mo NS AT Rs Vector Insert Vector Insert DNA Ligation high Ver 2 1 1 DNA Ligation high Ver 2 1 0 5 4 DNA Ligation high Ver 2 1 0 5 Vector Insert 1 3 100 4 3 pUC19 Hincll 50ng 25fmol HindIIlI
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