Pfu-X DNA polymerase (Solgent, South Korea) was used to amplify candidate TFs and promoters using Z. japonica leaf cDNA and genomic DNA as templates, respectively, using the corresponding primer sets (Supplementary Table 1
Pfu x dna polymerase
Manufactured by Solgent
Sourced in Cameroon
Pfu-X DNA polymerase is a thermostable DNA polymerase enzyme used for high-fidelity DNA amplification. It possesses 3'-5' exonuclease proofreading activity, enabling it to accurately replicate DNA with a low error rate.
Automatically generated - may contain errors
Lab products found in correlation
Gateway cloning technology, by Thermo Fisher Scientific (1 mentions)
Appropriate restriction enzymes, by Enzynomics (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Pgl3 control vector, by Promega (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taq polymerase, by Takara Bio (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Surecycler 8800 thermal cycler, by Agilent Technologies (1 mentions)
Pcrii topo vector, by Thermo Fisher Scientific (1 mentions)
9 protocols using pfu x dna polymerase
1
Protoplast-Mediated Transient Expression Assay
2
Cloning and Expression of Trichomonas vaginalis GP63
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To construct pET32a-GP63 expression plasmids, the coding sequence of the T. vaginalis GP63 (GenBank accession no. GU356538.1) were amplified by PCR using Pfu-X DNA Polymerase (Solgent, Daejeon, Korea) from cDNA of T. vaginalis T016 with a pair of oligonucleotide primers: GP63 forward, 5'-CGCGGATCCATGATATTGTCTCTACTCCTCGTTG-3' and reverse, 5'-CCCAAGCTTAATCGATAAATTTGCATCAGGTTCT-3' (recognition sites for BamHI and HindIII, respectively are underlined). The PCR products were digested with the appropriate restriction enzyme and cloned into the BamHI/HindIII sites of the pET-32a (+) expression vector with N-terminal His-tag. The resulting plasmids were named pET32a-GP63. Recombinant plasmids were confirmed by restriction analysis and PCR sequencing (Solgent, Daejeon, South Korea). His-tagged GP63 protein was induced by 1 mM IPTG at 37℃ for 3 hr in BL21 (DE3) Escherichia coli. Finally, GP63 proteins in the supernatant were separated by 10% SDS-PAGE and visualized by Coomassie blue staining or detected by western blotting using the anti-His antibody.
3
Cloning and Expressing Yeast PSAT Genes
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Full-length PSAT genes of S. cerevisiae (ScPSAT; NCBI ID: AJT97493.1) were acquired. The yeast PSAT-encoding genes were amplified by PCR using genomic DNA obtained from the Korean Collection for Type Cultures as a template, Pfu-X DNA polymerase (SolGent, Daejeon, Republic of Korea), and oligonucleotide primers (Cosmo Genetech Inc., Seoul, Republic of Korea). The amplified products were digested with appropriate restriction enzymes (Enzynomics, Daejeon, Republic of Korea) at 37 °C for 3 h. The digested products were ligated with the pET28a vector using T4 ligase (M0202S; Roche, Basel, Switzerland) at 18 °C overnight and subsequently transformed into E. coli strain DH5α. The transformants were confirmed by colony PCR and DNA sequencing.
4
Poplar Circadian Clock Genes
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The poplar GIGANTEA (PagGI) and ZEITLUPE (PagZTL) genes were identified by sequence comparison of Arabidopsis GI and ZTL, respectively, to the Populous trichocarpa genomic database. Poplar CO2 (PagCO2) and SOS2 (PagSOS2) were identified based on the cDNA sequences of PtCO2 and PtSOS2 respectively. The coding sequences of PagGIa, PagGIb, PagGIc, PagZTL1, PagZTL2, PagCO2 and PagSOS2 were amplified from cDNA from P. alba × P. glandulosa leaves with Pfu‐X DNA polymerase (Solgent, Daejeon, Korea) using the primer pairs listed in Table S1. GenBank accession numbers are shown in Table S2.
5
Construction of Reporter and Expression Vectors
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To construct pGL3-CIC 3′UTR, entire human CIC 3′UTR sequences (606nt) were amplified from cDNA of MCF7 cells using Pfu-X DNA polymerase (SolGent, Daejun, Republic of Korea) and cloned into the pGL3-control vector (Promega, WI, USA). To construct pGL3-CRABP1 pro, human CRABP1 promoter region (−1927bp ∼ +2bp) was amplified from MCF7 genomic DNA using Pfu-X DNA polymerase and cloned into the pGL3-basic vector (Promega, WI, USA). To make pHAGE-FLAG-CIC-S, and CIC-L, mouse Cic-S and Cic-L coding sequences were amplified from cDNA of NIH3T3 cells using Pfu-X DNA polymerase and cloned into the pHAGE-FLAG lentiviral vector. To make pHAGE-FLAG-CRABP1, human CRABP1 coding sequences were amplified from cDNA of MCF7 cells using Pfu-X DNA polymerase and cloned into the pHAGE-FLAG lentiviral vector.
6
Genetic Engineering of ICAM-1 Mutants
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The point mutant structure of the ICAM-1 tyrosine residue was generated using pfu-X DNA Polymerase (Solgent, SPX16-R250) and performed according to the manufacturer’s protocol. The mutant form of DNA was transformed with DH5α and the plasmid DNA sequence was analyzed using the Macrogen company’s DNA sequencing service. For the generation of deletion mutant of ICAM-1 domain, PCR was performed. In the first PCR step, an insert DNA was made, and was denatured at room temperature, and then the deleted DNA was amplified through the final PCR step. The insert and backbone DNAs were digested using the restriction enzyme, followed by ligation and transformation step. Primer sequence used for point mutations and deletion mutations are listed in Supplementary Table .
7
Bacterial DNA Extraction and Analysis
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Bacterial genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) and following the manufacturer’s instructions. Plasmid DNA was separated with the Dokdo-PrepTM Plasmid Mini-Prep Kit (Elpis-Biotech, Daejeon, South Korea). The concentration and the quality of the extracted DNA were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Polymerase chain reaction (PCR) was performed using Pfu-X DNA Polymerase (Solgent, Daejeon, South Korea) or Taq polymerase (TaKaRa, Kusatsushi, Japan). The PCR mixture and reaction conditions were maintained according to the manufacturer’s instructions using a Sure Cycler 8800 Thermal cycler (Agilent Technologies, Santa Clara, CA, USA). The list of primers and their targets used in this study are shown in Table 2 .
8
Genomic DNA extraction and guide sequence amplification
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To prepare genomic DNA, blastocysts were washed twice in phosphate-buffered saline (PBS)/PVA, treated with proteinase K, and incubated at 50°C for 3 h. Portions of genomic DNA containing guide sequences for sgRNAs (exon 2 or exon 5 of porcine OCT4) were amplified by PCR using the PCR primers listed in Table 1 and Pfu-x DNA polymerase (Solgent; Daejun, Korea). Resulting PCR products (~300 bp) were purified by gel purification, and PCR products were cloned into the pCR-II-Topo vector (Life Technologies; Foster City, CA, USA) or sequenced directly using the PCR primers used for amplification.
9
Site-Directed Mutagenesis of FbFP Gene
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Site directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) to introduce the single and double mutations into the EcFbFP gene. Briefly, the FbFP gene was amplified from pEcFbFP using Pfu-X DNA polymerase (Solgent, Daejeon, Korea), resulting in nicked circular strands of the plasmid. The plasmid was digested using DpnI to remove the residual template DNA and transformed into E. coli TOP10 cells by heat shock transformation at 42°C.
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