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 [ ] 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

2 100 020406080100120 Ligationhigh 1 B 1 LGK-201 2 [ ] 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)

3 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) [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.

4 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. 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 Insert = 1 3 100 3 1.

5 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. coli DH5 <100 L> LB/Amp(X-Gal IPTG)

6 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 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.

7 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. 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 [ ]

8 1/10 1/10 DNA DNA PCR PCR DNA DNA TA DNA TA APPEDIX PCR TA 2 16 2 DNA DNA -30 C LGK-201 8 [ ] APPENDIX ( g ) 80 ( L) 10 Buffer 10 E.

9 Coli Alkaline Phosphatase ( 1U/ L)* 10 Total 100 * 5 20pmoles 5 2U ( 10%) 3 60 C 60min 5 37 C 60min MagExtractor -PCR & Gel Clean up- (Code No.: NPK-601) (1)50pmole/ L( M) Primer (2) Primer (50pmole/ L [50 M]) 14 ( L) 10 Protruding End Kinase Buffer 2 10mM rATP <Code No. ATP-111 > 2 T4 Polynucleotide Kinase(5 20U/ L)*1 2 Total 20 37 C 1h 95 C 5min T4 Polynucleotide Kinase 50 L Q 10pmole/ L [10 M] primer *2*3 PCR *1 T4 Polynucleotide Kinase 10% *2 PCR *3 9 LGK-201 PCR Code No.

10 RATP 50 ATP-111 MagExtractor -Plasmid- < Plasmid DNA > 500 NPK-301 MagExtractor -PCR & Gel Clean up- < DNA fragment > 200 NPK-601 Magical Trapper < > MGS-101 Competent high DH5 < DH5 SOC > 100 L 10 DNA-903 Competent high JM109 < JM109 SOC > 100 L 10 DNA-900 KOD -Plus- PCR 200U 1 KOD-201 KOD -Plus- PCR 200U 1 KOD-211 KOD FX PCR 200U 1 KFX-101 (1) PCR PCR (2) PCR 1 g 35 ( L) 10 Blunt End Kinase Buffer 5 10mM rATP*1 <Code No. ATP-111 > 5 T4 Polynucleotide Kinase(5 20U/ L)*1 5 Total 50 37 C 1h *1 T4 Polynucleotide Kinase 10% ( ) 530-0001 TEL 06-6348-3786 FAX 06-6348-3833 E-mail : ( ) 104-8345 17 10 TEL 03-6887-8819 FAX 03-6887-8951 E-mail : TEL 06-6348-3888 FAX 06-6348-3833 9:00 12:00 , 13:00 17:00 ( ) E-mail : URL