KOD Plus Neo - bio-toyobo.cn

1 10-04 PCR kod plus - Neo (Code:KOD-401) TOYOBO CO., LTD. Life Science Department OSAKA JAPAN 1 .. 1 2 PCR .. 3 3 .. 6 4 .. 7 5 .. 9 6 .. 10 7 .. 11 8 .. 11 -20 C 1 1 KOD - plus - Neo Thermococcus kodakaraensis KOD1 KOD DNA Polymerase1,2) PCR KOD DNA Polymerase 3 5 Exonuclease Proofreading PCR KOD DNA Polymerase KOD - plus - Code No. KOD-201 KOD - plus - Code No. KOD-211 Taq 80 1 kb 30 kb PCR Polymerase 3 5 Exonuclease Hot start PCR KOD - plus - Neo * Taq 80 *KOD DNA polymerase PCR 20 30 < > < > 20mer Tm * 63 C 2 010203040 PCR KOD - plus -Neo 1 010203040 PCR KOD - plus -Neo 1 * Tm Nearest Neighbor method Tm 50 mM Na+ M Oligonucleotide Primer20mer 22mer Tm > 63 CTm 63 C2 3 * Step Down * Tm Primer20mer 22mer Tm > 63 CTm 63 C2 3 * Step Down * Tm ( )

2 021-58794900 2 1 kb 30 kb kb DNA Mg2+ 2 mM kb DNA KOD-401 (200 U 1) KOD-401B KOD - plus - Neo ( U/ l) 200 l 1 (KOD-401) 5 10 PCR Buffer for KOD - plus - Neo 1 ml 1 25 mM MgSO4 1 ml 1 2 mM dNTPs 1 ml 1 KOD - plus - Neo DNA -globin kb 3 2 PCR 1. 22~35mer Tm * 63 C GC 45~60% GC GC 3 5 60~70%, 3 40~50% 3 G C GC 3 25~35mer Tm 65 C 25mer Tm 65 C inosine acid * Tm Nearest Neighbor method Tm 50 mM Na+ M Oligonucleotide 2.

3 PCR Components Volume Final Concentration Autoclaved, distilled water (33 - X) l 10 PCR Buffer for KOD - plus - Neo 5 l 1 2 mM dNTPs 5 l mM each 25 mM MgSO4 3 l mM (10 M each) l M each X l Genomic DNA ~200 ng/50 l Plasmid DNA ~50 ng/50 l cDNA ~200 ng/50 l KOD - plus - Neo (1 U/ l) 1 l 1 U/50 l Total 50 l Vortex PCR Mg2+ mM Buffer RNA PCR PCR 50 l RNA 50 ng Total RNA1 g 20 l 1 l 3 1 ( ) 021-58794900 4 3. PCR [ ] KOD - plus - Neo Tm Tm 63 C 68 C 1) Tm 63 C Predenature : 94 C, 2 min. Denature : 98 C, 10 sec. Extension : 68 C, 30 kb 30~45 Extension 30 kb 10 kb 1 kb Mg2+ 2 mM DNA Denature 94 C 15 sec.

4 GC rich 98 C 10 sec. DMSO 25~35mer Tm 68 C Tm 68 C DMSO 2% 25 45 cycles Primer20mer 22mer Tm > 63 CTm 63 C2 3 * Step Down * Tm Primer20mer 22mer Tm > 63 CTm 63 C2 3 * Step Down * Tm 5 2) Tm 63 C Step down Predenature : 94 C, 2 min. Denature : 98 C, 10 sec. Annealing : (Tm) C, 30 sec. Extension : 68 C, 30 kb Tm Step down Predenature : 94 C, 2 min. Denature : 98 C, 10 sec. Extension : 74 C, 30 kb Denature : 98 C, 10 sec. Extension : 72 C, 30 kb Denature : 98 C, 10 sec. Extension : 70 C, 30 kb Denature : 98 C, 10 sec. Extension : 68 C, 30 kb Extension : 68 C, 7 min. Step down 10 kb 1 kb Mg2+ 2 mM DNA Denature 94 C 15 sec.

5 GC rich 98 C 10 sec. 15 30 cycles 5 cycles 5 cycles 5 cycles 25 45 cycles ( ) 021-58794900 6 3 1. PCR PCR 50 l Genomic DNA DNA 5~200 ng 50 ng DNA ~100 ng 10 ng Plasmid DNA 10 pg~50 ng 1 ng cDNA ~200 ng (RNA ) 50 ng (RNA ) DNA 10 pg~10 ng 1 ng PCR RNA PCR RNA PCR 50 l PCR 50 ng RNA Total RNA 1 g 20 l 1 l GC rich 2~5% DMSO DMSO DMSO 25~35mer Tm 68 C Tm 68 C DMSO 2% PCR total 50 l dNTPs dNTPs Mixture(2mM) (Code No.:NTP-201) 2.

6 PCR PCR PCR PCR TA KOD DNA Polymerase TA TArget Clone - plus - Code No. TAK-201 PCR TA PCR DNA Polymerase 3 5 Exonuclease / DNA MagExtractor -PCR & Gel Clean up- Code No. NPK-601 7 4 1. DNA -globin kb PCR TArget Clone - plus - Code No. TAK-201 TA 96 KOD - plus - Neo KOD - plus - KOD - plus - Taq DNA Polymerase 80 PCR 2. KOD - plus - Neo PCR DNA -globin PCR 30 KOD - plus - Neo kb kb KOD - plus - Neo DNA PolymeraseA Long PCR Pfu DNA PolymeraseKOD FXKOD - plus -KOD - plus -Ver.

7 2 KOD - plus -Neo Mutation frequency 100,000 PCR DNA PolymeraseA Long PCR Pfu DNA PolymeraseKOD FXKOD - plus -KOD - plus -Ver. 2 KOD - plus -Neo Mutation frequency 100,000 PCR M 1 2 3 4 5 1 2 3 4 5 1 2 34 5 1 2 3 4 5 KOD - plus -Ver. 2 KOD - plus -NeoA B 1: Human -globin( kb) 2: Human -globin( kb)3: Human -globin( kb) 4: Human -globin( kb) 5: Human -globin( kb)M: 1 kb DNA Ladder KOD - plus -Neo 1 4:94 C, 2 C, 10 C, 30 kb5:Step down 30 cycles< >30 kb 1 kb 1 kb kb M 1 2 3 4 5 1 2 3 4 5 1 2 34 5 1 2 3 4 5 KOD - plus -Ver. 2 KOD - plus -NeoA B 1: Human -globin( kb) 2: Human -globin( kb)3: Human -globin( kb) 4: Human -globin( kb) 5: Human -globin( kb)M: 1 kb DNA Ladder KOD - plus -Neo 1 4:94 C, 2 C, 10 C, 30 kb5:Step down 30 cycles< >30 kb 1 kb 1 kb kb ( ) 021-58794900 8 3.

8 HeLa Total RNA cDNA Total RNA ng 4 PCR 40 kod plus - Neo 4 4. DNA 50 ng -globin kb KOD - plus - Neo KOD - plus - Neo 30 kb M1234 M 1234 KOD - plus -Neo 94 C, 2 C, 10 C, X30 cycles X:1: 36 sec. (10 kb)2: 54 sec. (15 kb)3: 108 sec. (30 kb)4: 216 sec. (60 kb) kbM: 1 kb DNA LadderM1234 M 1234 KOD - plus -Neo 94 C, 2 C, 10 C, X30 cycles 94 C, 2 C, 10 C, X30 cycles X:1: 36 sec. (10 kb)2: 54 sec. (15 kb)3: 108 sec. (30 kb)4: 216 sec. (60 kb) kbM: 1 kb DNA LadderM 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 KOD - plus -NeoA B KOD - plus -Ver. 2 KOD - plus -Neo 94 C, 2 C, 10 C, 30 kb40 cycles< >30 kb 1 kb 1 kb 30 kb 1: Human transferrinreceptor ( kb)2: Human Tuberous scleosis( kb)3: Human adaptin( kb)4: Human polymerase ( kb)M: 1 kb DNA LadderM 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 KOD - plus -NeoA B KOD - plus -Ver.

9 2 KOD - plus -Neo 94 C, 2 C, 10 C, 30 kb40 cycles< >30 kb 1 kb 1 kb 30 kb 1: Human transferrinreceptor ( kb)2: Human Tuberous scleosis( kb)3: Human adaptin( kb)4: Human polymerase ( kb)M: 1 kb DNA Ladder 9 5 1 Total RNA ORF HeLa Total RNA cDNA Total RNA 50 ng ORF open reading frame PCR 30 KOD - plus - Neo 2 DNA 200 ng tPA Human tissue-type plasminogen activator kb Mg2+ 2 mM 10 kb Mg2+ 2 mM M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 KOD - plus -NeoA B 1: Human brkTyrosine kinase( kb) 2: Human racprotein kinase-alpha ( kb)3: Human 63kDa protein kinase( kb)4: Human c-synprotooncogene( kb) 5: Human FER Tyrosine kinase( kb) 6: Human cell adhesion kinasebeta ( kb) 7: Human Jak2 kinase( kb)M: 1 kb DNA Ladder< >30 kb 1 kb 30 kb KOD - plus -Neo 94 C, 2 C, 10 C, 30 kb30 cyclesM 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 KOD - plus -NeoA B 1: Human brkTyrosine kinase( kb) 2: Human racprotein kinase-alpha ( kb)3: Human 63kDa protein kinase( kb)4: Human c-synprotooncogene( kb) 5: Human FER Tyrosine kinase( kb) 6: Human cell adhesion kinasebeta ( kb) 7.

10 Human Jak2 kinase( kb)M: 1 kb DNA Ladder< >30 kb 1 kb 30 kb KOD - plus -Neo 94 C, 2 C, 10 C, 30 kbM 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 M 1 2 3 4 5 6 7 KOD - plus -NeoA B 1: Human brkTyrosine kinase( kb) 2: Human racprotein kinase-alpha ( kb)3: Human 63kDa protein kinase( kb)4: Human c-synprotooncogene( kb) 5: Human FER Tyrosine kinase( kb) 6: Human cell adhesion kinasebeta ( kb) 7: Human Jak2 kinase( kb)M: 1 kb DNA Ladder< >30 kb 1 kb 30 kb KOD - plus -Neo 94 C, 2 C, 10 C, 30 kb30 cyclesM 1 2 3 KOD - plus -Neo Step down cycle1: 30 kb Mg mM 2: 1 kb Mg mM 3: 30 kb Mg 2 mM kbM: /HindIIIdigestM 1 2 3 KOD - plus -Neo Step down cycle1: 30 kb Mg mM 2: 1 kb Mg mM 3: 30 kb Mg 2 mM kbM: /HindIIIdigest ( ) 021-58794900 10 6 1 kb 2~5 Tm-5~Tm-10 C Step down 10 kb MgSO4 mM mM 10 kb GC rich mM mM RNA RNA cDNA RNA 1 U ~ U 2~5% DMSO [ 5.]