Transcription of Ligation high Ver - TOYOBO
1 22-05 - DNA Ligation Kit - Ligation high (Code No. LGK-201) TOYOBO CO., LTD. Bioproducts Sales and Marketing Department OSAKA JAPAN A3706K [ ] .. 1 [ ] .. 2 [ ] .. 3 [ ] .. 4 .. 4 .. 4 .. 5 TA .. 5 TA PCR .. 6 .. 6 [ ] .. 7 [ ] APPENDIX .. 8 .. 8 .. 8 .. 8 PCR .. 9 .. 9 1 LGK-201 [ ]
2 DNA T4 DNA Ligase DNA 1 Ligation high Ligation high Ligation high T4 DNA Ligase Ligation high TA -20 C Ready-to-use Ligation high 1 DNA 1 TA Ligationhigh 100 020406080100120 Ligationhigh 1 B 1 1 TA Ligationhigh 100 020406080100120 Ligationhigh 1 B 1 LGK-201 2 [ ]
3 Ligation high 750 L 1 1 L 100 -20 C -30 C 2 TA 0 100 02040608010012005 2 TA 0 100 02040608010012005 3 LGK-201 [ ] DNA *1 [4] TE Buffer (10mM Tris-HCl[ ], 1mM EDTA)
4 DNA *2 TA *3 2 2 5 *4 4 C 25 C 16 C *5 10% 1 Ligation high DNA Ligation 1 1 or 1 16 C 5 30 1 1 or 1 16 C 30 1 1 or 1 16 C 30 T PCR 1 1 16 C 30 T *1*2 PCR 1 1 16 C 30 1 1 or 1 16 C 30 *1 *2 1 L X PCR 1 Ligation high 15 ( L)
5 [p6] DNA DNA*1 L Ligation high L*2 16 C 30 *3*4 100 L 10 L *5 LGK-201 4 [ ] pUC19/HindIII 50ng, 25fmol DNA HindIII 564bp DNA < L> Ligation high < L> 16 C 30 10 L E. coli DH5 <100 L> LB/Amp(X-Gal IPTG) Vector Insert 1 3 pUC19/HincII 50ng, 25fmol PCR KOD -Plus- DNA *1 DNA < L> Ligation high < L or L> 16 C 30 10 L E.
6 Coli DH5 LB/Amp(X-Gal IPTG) Vector Insert 1 3 *1 KOD -Plus- DNA 0204060801001201 11 31 5 0204060801001201 11 31 5 Vector Insert Vector Insert DNA Ligationhigh = 1 1 DNA Ligationhigh = 1 4 DNA Ligationhigh = 1 Vector Insert = 1 3 100 0204060801001201 11 31 5 0204060801001201 11 31 5 Vector Insert Vector Insert DNA Ligationhigh = 1 1 DNA Ligationhigh = 1 4 DNA Ligationhigh = 1 Vector
7 Insert = 1 3 100 3 1:5 100 0204060801001201 11 31 5 Vector Insert 3 1:5 100 0204060801001201 11 31 5 Vector Insert 5 LGK-201 pUC19/HincII 50ng, 25fmol HindIII 10mer DNA < L> Ligation high < L or L> 16 C 30 10 L E. coli DH5 LB/Amp(X-Gal IPTG) Vector Linker 1 10 Ligation high 1/2 DNA Ligation high = 1 TA T 50ng, 25fmol PCR Taq DNA polymerase DNA < L> Ligation high < L > 16 C 30 10 L E.
8 Coli DH5 <100 L> LB/Amp(X-Gal IPTG) Vector Insert 1 3 Vector Insert Vector Insert DNA Ligationhigh = 1 1 DNA Ligationhigh = 1 5 DNA Ligationhigh = 1 Vector Insert = 1 10 100 0204060801001201 11 31 51 10 0204060801001201 11 31 51 10 Vector Insert Vector Insert DNA Ligationhigh = 1 1 DNA Ligationhigh = 1 5 DNA Ligationhigh = 1 Vector Insert = 1 10 100 0204060801001201 11 31 51 10 0204060801001201
9 11 31 51 10 Vector Insert 6 TA 1:5 100 0204060801001201 11 31 5 Vector Insert 6 TA 1:5 100 0204060801001201 11 31 5 LGK-201 6 TA PCR T 50ng, 25fmol PCR Taq DNA polymerase DNA < L> Ligation high < L > 16 C 30 10 L E. coli DH5 <100 L> LB/Amp(X-Gal IPTG) PCR DNA PCR EcoRI ZAP II(500ng) pRheo/EcoRI test insert 225ng 5 L 5 L L 16 C 30min L GIGAPACK III Stratagene E.
10 Coli XL-1 Blue MRF Ligation high Ligation high Ligation high 2 2 Ligation high Ligation high Ligation high DNA Ligation 1 1 1 1 (pfu/ g ZAP II) 106 106 106 PCR l <15 l > 7 TA l 100 PCR l <15 l > 7 TA l 100 7 LGK-201 [ ] 1/10 1/10 DNA DNA PCR PCR DNA DNA TA DNA TA APPEDIX PCR TA 2 16 2 DNA
