Restriction enzyme cloning was accomplished using: BsaI-HFv2 (New England Biolabs, #R3733), Esp3I (New England Biolabs, R0734) and T4 Ligase (Promega, M1801). PCR was performed using proofreading enzymes iProof High-Fidelity DNA Polymerase (Biorad, #1725301), Phusion High-Fidelity DNA Polymerase (New England Biolabs, M0530), or Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491). Molecular biology experiments, including in silico designs and experimentation, as well as plasmid maps, were designed using SnapGene software (https://www.snapgene.com:443/products/snapgene/ ) (GSL Biotech LLC). All plasmids developed in this project are listed in Tables S1 and S2 and the Key resources table .
Iproof high fidelity dna polymerase
Manufactured by Bio-Rad
Sourced in United States, Sweden, Germany
The iProof High-Fidelity DNA Polymerase is a thermostable DNA polymerase enzyme designed for high-accuracy DNA amplification. It features enhanced fidelity and processivity, enabling efficient and accurate DNA synthesis for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (4 mentions)
Xhoi, by New England Biolabs (4 mentions)
Bamhi, by New England Biolabs (4 mentions)
Qiaprep spin miniprep kit, by Qiagen (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Pcr blunt 2 topo vector, by Thermo Fisher Scientific (3 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Bp clonase 2 enzyme mix, by Thermo Fisher Scientific (3 mentions)
Nucleospin plasmid kit, by Macherey-Nagel (3 mentions)
Bsai hfv2, by New England Biolabs (2 mentions)
Esp3i, by New England Biolabs (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Snapgene software, by GSL Biotech (2 mentions)
T4 ligase, by Promega (2 mentions)
T4 dna ligase, by Roche (2 mentions)
Restriction enzyme, by New England Biolabs (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Pqe30, by Qiagen (2 mentions)
Pgem t vector, by Promega (2 mentions)
96 protocols using iproof high fidelity dna polymerase
1
Molecular Cloning and PCR Techniques
2
Amplification and Sequencing of Rider Transposon
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Primers located in the 5′UTR and first intron of the J-2 locus were used to amplify the DNA segment containing the Rider transposon. Amplification time was set up at 4 min at 72 °C using j-2 (LA3899) DNA as matrix and the iProof High-Fidelity DNA Polymerase (Bio-Rad). Amplified fragment was subsequently cloned into the pGEM-T Easy vector (Promega) and subjected to sequence analysis. Sequences were annotated using blast search against the SOL Genomics Network (SGN; https://solgenomics.net/ ).
3
Cloning and Analyzing VEGF-A Promoter Activity
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For the reporter constructs, the VEGF-A promoter regions (−2,000 to +50 bp) were amplified from human genomic DNA (Zyagen, San Diego, CA, USA) by PCR using iProof™ High-Fidelity DNA Polymerase (Bio-Rad Laboratories, Inc.) (23 (link)). The PCR products were subcloned into the pGL4.11[luc2P] vector (Promega Corporation, Madison, WI, USA) upstream of a luciferase gene using appropriate restriction sites. Putative transcription factor binding sites were identified with the Transcription Element Search System (TESS) algorithm (http://www.cbil.upenn.edu/downloads/ ).
The transcription factor binding to the VEGF-A promoter was analyzed by luciferase assay as previously reported (20 (link),23 (link)). Briefly, MCF-7 cells (1×105) were transfected in 24-well plates with 1 µg pGL4.11[luc2P] luciferase reporter vector driven by the VEGF-A promoter, or with 2 µg control vector. For transfection of MCF-7 cells, 0.2×105 cells/well were seeded into a 24-well plate and grown for 24–48 h. Cells were harvested 48 h post-transfection, and efficiency was measured by qPCR, immunostaining and western blotting.
The transcription factor binding to the VEGF-A promoter was analyzed by luciferase assay as previously reported (20 (link),23 (link)). Briefly, MCF-7 cells (1×105) were transfected in 24-well plates with 1 µg pGL4.11[luc2P] luciferase reporter vector driven by the VEGF-A promoter, or with 2 µg control vector. For transfection of MCF-7 cells, 0.2×105 cells/well were seeded into a 24-well plate and grown for 24–48 h. Cells were harvested 48 h post-transfection, and efficiency was measured by qPCR, immunostaining and western blotting.
4
Generating Transgenic Arabidopsis Plants
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To generate overexpression constructs, complete open reading frame (ORF) of OsPP108 was PCR amplified using iProof high-fidelity DNA polymerase (Bio-Rad, Hercules, USA) and cloned at BamHI and SalI restriction sites of modified pCAMBIA1300 vector under the control of CaMV35S promoter. The binary construct was transferred to Agrobacterium tumifaciens strain GV3101 and used for Arabidopsis plant transformation by floral dip method [27 (link)]. T0 seeds harvested from these plants were screened on selection media (half strength MS media supplemented with 15μg/ml hygromycin) to obtain T1 plants. Transgenic plants were verified by segregation analysis in selection media (15μg/ml hygromycin) and then transferred to soil till maturity, to generate T2 and T3 generation, which were screened as homozygous transformants and used for further analysis.
5
Cloning Promoter Deletion Constructs
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To generate promoter deletion constructs, primary monocyte genomic DNA was extracted from the interphase and phenol layer using TRIzol reagent (Life Technologies), according to the manufacturer's instructions. To obtain 5′ end promoter deletion products, primers (Supplementary Table S1 ) flanking the desired promoter regions were used for PCR amplification, using the purified genomic DNA as template. PCR was performed using iProof High Fidelity DNA Polymerase (Bio-rad) under the following parameters: initial denaturation at 98°C; 40 cycles of amplification, denaturation at 98°C for 10 s, annealing at Tm of primer pair +3°C for 30 s, elongation at 72°C for 2.5 min; followed by a final extension at 72°C.
The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were XhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).
The isolated promoter lengths were separately cloned into pGL4.20 vector (Promega) using standard molecular cloning techniques. The restriction enzymes used were XhoI and HindIII (Thermo Fisher Scientific). T4 DNA ligase was from Roche. For small-scale purification of plasmids, AxyPrep Plasmid Miniprep kit (Axygen Biosciences) was used. Large-scale purification of plasmids intended for transfection was carried out using PureLink HiPure Plasmid Filter Purification kit (Invitrogen). The full-length sequence of each promoter construct was confirmed by sequencing using Big Dye Terminator cycle sequencing kit and ABI Prism 3100 Genetic Analyzer (Life Technologies).
6
Plasmid DNA Extraction and Manipulation
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Plasmid DNA was extracted from E. coli with the GenEluteTMHP Plasmid miniprep purification kit, as recommended by the manufacturer (Sigma). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs and Promega, respectively). Oligonucleotide primers were synthesized by Eurogentec (Seraing, Belgium). PCR was performed in a T100 thermal cycler (Biorad) with the iProof high-fidelity DNA polymerase (Biorad). Amplified DNA fragments were purified with a PCR purification kit (Roche) and separated by electrophoresis in 1% agarose gels after digestion. Hydrolyzed DNA fragments were extracted from agarose gels with the NucleoTrap kit from Macherey-Nagel. All constructs were confirmed by DNA sequencing (MWG).
7
Reverse Transcription and S Gene Sequencing of MERS-CoV
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Total RNA from nasal turbinate tissue samples from dromedary camels and alpaca were extracted using the RNeasy Mini Kit (Qiagen) and cDNAs were synthesized using random hexamers and the High Capacity RNA to cDNA Kit (Thermo Fisher, Waltham, MA, USA). cDNA was subsequently used to PCR-amplify the MERS-CoV S using iProof High-Fidelity DNA Polymerase (Biorad, Hercules, CA, USA) according to the manufacturer’s protocol; primer sequences are available upon request. Sequences were assembled on SeqMan Pro (DNASTAR, Madison, WI, USA) and analyzed on MegAlign (DNASTAR, Madison, WI, USA) by comparison to the MERS-CoV (strain HCoV-EMC/2012) input sequence.
8
Recombinant Expression and Purification of Arabidopsis Thioredoxins
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For the expression of Arabidopsis GPX1 and GPX7, the corresponding cDNAs, excluding the predicted transit peptides and the stop codons, were amplified with iProof™ High-Fidelity DNA Polymerase (Bio-Rad) using oligonucleotides listed in Table S1 , which added NcoI and XhoI sites at the 5’ and 3’ ends, respectively. For the expression of Arabidopsis TRX m4, the corresponding cDNA, excluding the predicted transit peptides and including the stop codon, was amplified as described above using oligonucleotides (Table S1 ) adding BamHI and HindIII sites at the 5’ and 3’ ends, respectively. PCR products were gel-purified, cloned in pGEMt vector (Promega), and sequenced. For GPXs, pGEMt-derived plasmids were digested with NcoI and XhoI, subcloned in the pET28 (Qiagen) expression vector and introduced into E. coli BL21 (DE3) cells. The pGEMt-TRX m4 plasmid was digested with BamHI and HindIII, subcloned in the pQE30 (Qiagen) expression vector and introduced into E. coli XL1Blue. Over-expressed recombinant proteins, containing a His-tag at the C-terminus (GPX1 and GPX7) or the N-terminus (TRX m4), were purified by nitrile triacetic acid (NTA) affinity chromatography in Hi-Trap affinity columns (GE Healthcare). Recombinant His-tagged NTRC and 2-Cys PRX from rice [38 (link)] and TRXs of the types y2, x and f2 from Arabidopsis were obtained as previously described [31 (link),41 (link)].
9
Constructing Knockout Mutants of S. mutans
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To construct spaP and cnm knockout mutants in S. mutans wild type strain 49 (no. 23 in Fig. 2 , Fig. S2), a PCR overlapping strategy was used involving insertion of a selective marker (ermAD or add9) within the target gene. The DNA was purified using GeneElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Sweden), PCR amplifications were done with iProof High fidelity DNA polymerase (Bio-Rad, Sweden) and all primers (Table S2) were from Eurofins Genomics (Germany). Briefly, the 5′- and 3′-regions of the target gene were fused to the selective marker by end homology PCR and sub cloned into ZeroBlunt TOPO vector and transformed into a TOPO10 E. coli strain (Bio-Rad, Sweden). Correct generated disruption fragments were confirmed by sequencing (Eurofins Genomics, Germany). For construction of knockout mutants the cloned disruption fragments were amplified using T7 and Sp6 primers, transformed into isolates of S. mutans strain 49 by using a competence-stimulating peptide (SGSLSTFFRLFNRSFTQALGK) (Li et al., 2001 ). Correctly integrated fragments were confirmed by PCR and sequencing (Eurofins Genomics, Germany) (see also Fig. S5).
10
CRISPR sgRNA Vector Construction
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We replaced the unc-119 target sequence in pU6::unc-119 sgRNA vector (Friedland et al. 2013 (link)) with the desired target sequence using overlap extension PCR. The pU6::unc-119 sgRNA vector was diluted to 2 ng/µl and used as a template to generate two overlapping fragments. The first was amplified using the primers CMo16428 and sgRNA R, resulting in the U6 promoter fused to the GN19 target sequence (U6p::GN19). The second was amplified using the primers CMo16429 and sgRNA F, resulting in the GN19 target sequence fused to the sgRNA scaffold and U6 3′-UTR. These two PCR products were mixed together, diluted 1:50, and used as a template for a PCR reaction with primers CMo16428 and CMo16429. The resulting pU6::target sequence::sgRNA scaffold::U6 3′-UTR fusion products were gel purified and inserted into the pCR-Blunt II-TOPO vector (Invitrogen, no. K2800-20). We used iProof high-fidelity DNA polymerase (Bio-Rad, no. 172-5300) in all PCR reactions above to minimize errors of PCR amplification, and all the constructs were confirmed by DNA sequencing. Primers sequences are listed in Table S2 .
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