The VvPDS exon was amplified from genomic DNA of both cultivars using High-Fidelity DNA polymerase KOD-plus Neo (TOYOBO, Japan). Specific primers (Supplementary Table S1 ) were designed based on the homologous gene VIT_09s0002g00100 from the EnsemblPlants1 . Thermocycling conditions were as follows: 95°C for 5 min; 45 cycles of 95°C for 10 s, 57°C for 30 s, and 68°C for 30 s; followed by a final extension at 68°C for 5 min. The PCR product was cloned into the pClone007 Simple Vector (TSINGKE), and around five clones were sequenced.
High fidelity dna polymerase kod plus neo
Manufactured by Toyobo
Sourced in Japan
The High-Fidelity DNA polymerase KOD-plus Neo is a DNA polymerase enzyme used in various molecular biology applications. It has high fidelity, which means it can accurately replicate DNA with a low error rate. The enzyme is derived from the thermophilic archaeon Thermococcus kodakarensis.
Automatically generated - may contain errors
5 protocols using high fidelity dna polymerase kod plus neo
1
Genotypic Characterization of VvPDS Exon
2
Genetic Engineering of Streptomyces sp. 4F
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E. coli strain DH5α was used for cloning of plasmids with pUC replication origin (pUCori); strain EPI300 (Epicentre) was used for cloning oriV-containing plasmids; and strain ET12567 was used for conjugation with Streptomyces sp. 4F [26] (link). E. coli strains were grown in Luria-Bertani medium [27] (link), while Streptomyces sp. 4F was grown on mannitol soya flour [28] (link) plates for spore preparation and on R2YE plates [29] for actinorhodin production.
Enzymes used in this study (unless specified) were purchased from NEB. GeneRuler 1-kb DNA ladder was purchased from Thermo Fermentas and λ-HindIII ladder was from TAKARA. High fidelity DNA polymerase KOD Plus Neo was purchased from TOYOBO. The lycopene standard was purchased from Sigma-Aldrich.
Primers used in this study were listed inTable S1
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E. coli strain DH5α was used for cloning of plasmids with pUC replication origin (pUCori); strain EPI300 (Epicentre) was used for cloning oriV-containing plasmids; and strain ET12567 was used for conjugation with Streptomyces sp. 4F [26] (link). E. coli strains were grown in Luria-Bertani medium [27] (link), while Streptomyces sp. 4F was grown on mannitol soya flour [28] (link) plates for spore preparation and on R2YE plates [29] for actinorhodin production.
Enzymes used in this study (unless specified) were purchased from NEB. GeneRuler 1-kb DNA ladder was purchased from Thermo Fermentas and λ-HindIII ladder was from TAKARA. High fidelity DNA polymerase KOD Plus Neo was purchased from TOYOBO. The lycopene standard was purchased from Sigma-Aldrich.
Primers used in this study were listed in
3
Amplification and Sequencing of Hygromycin-Resistance Gene
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Specific primers for the hygromycin-resistance gene (Supplementary Table S1 ) were used for PCR with High-Fidelity DNA polymerase KOD-plus Neo (TOYOBO, Japan). The thermocycler was set at 95°C for 5 min; 34 cycles of 95°C for 10 s, 60°C for 30 s, and 68°C for 30 s; followed by a final extension at 68°C for 5 min. Amplicons were separated on an EtBr-stained agarose gel (1.0%), then cloned into the pclone007 vector for Sanger sequencing.
4
Molecular Cloning of Viral Proteins
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TGMV cC4 was cloned into the pCHF3-GFP, pCHF3-CFP, pCHF3-Myc, p2YN, pGBKT7, and pMBP vectors. TGMV mC4 was cloned into the pCHF3-GFP, pCHF3-Myc, and p2YN vectors. TGMV mC4G2A was cloned into the pCHF3-GFP, p2YN, and pGBKT7 vectors. TLCYnV C4 was cloned into the pCHF3-GFP vector. NbToc64-III was cloned into the p2YN, pCHF3-Myc, pGADT7, and pGEX4T-3 vectors. NbPUB4 was cloned into the pCambia-FLAG, p2YC, pGEX4T-3, pET-32a, and pGADT7 vectors. AtMBS1 and NbMBS1 were cloned into the pCHF3-GFP, pGEX4T-3, and pCV vectors. TGMV cC4A20P, C4F41P, and C4E75P were cloned into the pCHF3-GFP, pCHF3-CFP, and pGBKT7 vectors, respectively. All cDNAs were PCR amplified using KOD-Plus-Neo High-Fidelity DNA Polymerase (TOYOBO, Japan). The resulting PCR fragments were cloned into the destination vectors using the One-Step Cloning Kit (Vazyme, China). TGMV cC4A20P, C4F41P, and C4E75P were generated by overlapping PCR methods. All primers used for plasmid construction are listed in Supplemental Table 4 .
5
Cloning and Mutagenesis of DRM2 and C4 Proteins
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NbDRM2 was cloned into p2YN, pGADT7, pGD-Flag, pGex4T-3, and pGD-GFP vectors, and TLCYnV C4 was cloned into p2YC, pGBKT7, pCHF3, pCHF3-CFP, pGex4T-3 and pgR106 vectors. C4(G2A), C4(P32A), C4(P33A), C4(N34A), C4(T35A), C4(T36A), C4(T38A), C4(S39A), C4(S43A), C4(T47A), C4(S49A), C4(T51A), C4(T55A), C4(T59A), and C4(T60A) were cloned individually into pGBKT7. C4(T35A) and C4(S43A) were cloned individually into pgR106 and pCHF3-CFP vectors. The part of the coding sequence of NbDRM2 was cloned into pTRV-RNA2 for VIGS. All cDNAs were PCR-amplified using the KOD-Plus-Neo High-Fidelity DNA polymerase (TOYOBO). The resulting PCR fragments were first cloned individually into the pMD18-T vector (TaKaRa), then released from the vector using corresponding restriction enzymes. The released fragments were ligased into the expression vectors. C4(P32A), C4(P33A), C4(N34A), C4(T35A), C4(T36A), C4(T38A), C4(S39A), C4(S43A), C4(T47A), C4(S49A), C4(T51A), C4(T55A), C4(T59A), and C4(T60A) mutants were generated by the overlapping PCR methods. All primers used for plasmids construction are listed in Supplemental Table 1 (S1 Table ).
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