Phusion dna polymerase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Finland, Germany, United Kingdom, China

Phusion DNA polymerase is a high-fidelity DNA polymerase used for PCR amplification. It possesses 3' to 5' exonuclease activity, which enables efficient proofreading and results in a low error rate during DNA synthesis.

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Phusion polymerase (407 citations) Phusion Hot Start II DNA Polymerase (91 citations) DNA polymerase (71 citations) Phusion Hot Start II High-Fidelity DNA Polymerase kit (4 citations) Phusion Flash High-Fidelity DNA Polymerase (4 citations) Phusion Hot Start II DNA polymerase kit (3 citations) Phusion DNA Polymerase kit (2 citations) Phusion (Pfu) DNA polymerase (2 citations) PHUSION II high-fidelity DNA polymerase (2 citations) Phusion proof-read DNA polymerase (2 citations)

487 protocols using phusion dna polymerase

Construction and Mutagenesis of IRF2BP1/2 Fusion Proteins

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mRNA was extracted from HeLa cells using the NucleoSpin^® RNA II kit (Macherey‐Nagel) according to the manufacturer's instructions. Following this, mRNA was transcribed into cDNA using the SuperScriptTM First‐Strand Synthesis System (Invitrogen). IRF2BP1 and IRF2BP2 open reading frames were amplified by PCR using gene‐specific primers, which also contained the restriction sites BamHI and XhoI and Phusion DNA Polymerase (Thermo Scientific). As a vector backbone, a modified pcDNA3.1 vector was used that already contained an N‐terminal HA‐tag. IRF2BP1 and IRF2BP2 PCR fragments were cloned into this vector using BamHI and XhoI restriction sites. Lysine to arginine mutations were introduced using the QuickChange^® Site‐directed Mutagenesis Kit (Stratagene) and site‐specific mutagenesis primers.
For generation of bi‐cistronic IRF2BP1/GFP constructs, PCRs were performed from HA‐IRF2BP1 constructs using gene‐specific primers, which also contained the restriction sites BamHI and EcoRI, and Phusion DNA Polymerase (Thermo Scientific). As a vector backbone, pIRES‐hrGFPII was used. IRF2BP1 PCR fragments were cloned into this vector using BamHI and EcoRI restriction sites. Site‐directed mutagenesis was done to generate constructs that are resistant to siRNA#5.
Sequencing of all constructs was done at GATC using a vector‐specific CMV primer as well as internal sequencing primers.

Barysch S.V., Stankovic‐Valentin N., Miedema T., Karaca S., Doppel J., Nait Achour T., Vasudeva A., Wolf L., Sticht C., Urlaub H, & Melchior F. (2021). Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling. EMBO Reports, 22(3), e49651.

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Recombinant NT-mini and Agrin Constructs

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Recombinant constructs for NT-mini (Gly497-Leu875) and C-terminal Agrin substrates (Pro1635-Pro2067) were amplified using a Phusion DNA polymerase (Thermo Fisher Scientific) starting from a source synthetic gene (GeneWiz) corresponding the full-length human NT cDNA (UniProt P56730) and from a human Agrin y0z0 cDNA (UniProt O00468) obtained from Source Biosciences, respectively, using the oligonucleotides listed in Suppl.
Table 1.
The resulting PCR products were subcloned into pCR4-TOPO vectors (Thermo Fisher Scientific) using BamHI and NotI restriction sites. These plasmids served as the basis for the generation of the inactivated Ser-825-Ala NT*-mini and of the various Agrin splice variants via site directed mutagenesis. The corresponding PCR reactions were performed with Phusion DNA polymerase (Thermo Fisher Scientific) using the oligonucleotides listed in Suppl. Table 1.
All constructs were verified using Sanger sequencing (Microsynth) and then were sub-cloned into modified pUPE.106.08 plasmids (U-Protein Express BV) for secreted protein expression in mammalian cell culture systems. For NT-mini, the final expression constructs contained an N-terminal 8xHis-SUMO tag used to stimulate expression and facilitate purification. For Agrin constructs, the final expression construct contained an N-terminal 6xHis-tag followed by a Tobacco Etch Virus Protease (TEV) cleavage site.

Canciani A., Capitanio C., Stanga S., Faravelli S., Kienlen‐Campard P., & Forneris F. (2020). NT-mini, a recombinant tool for the study of Neurotrypsin functionality.

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Lentiviral and Retroviral Vector Production

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For viral production, 10 cm dishes of HEK293T cells with a confluency of 60–80% were transfected with 15 μg transfer plasmid, 4.5 μg envelope plasmid pMD2.G and 10.5 μg packaging plasmid psPAX2 (lentivirus) or pCIG3.NG (retrovirus) using calcium phosphate-based transfection. Virus-containing medium was replaced with new medium at 16, 48 and 72 h and collected after 48 and 72 h. Cell debris was removed by centrifugation at 200× g for 5 min at room temperature, and the medium was stored at 4 °C until the final harvest. All harvested media were pooled, filtered with a 0.45-μm Falcon filter and concentrated 100x using an Amicon-15 100-kDa filter column (Merck, Z740210). Aliquots containing 20% of the concentrated viral supernatant were stored at -80 °C. For transduction, 100,000 cells seeded the previous day in 6well plates were transduced with the contents of one aliquot. Since the transfer plasmids encode a puromycin resistance gene, cells were selected after 96 h with 1 μg/ml puromycin to obtain stable cell lines. Point mutants of integrin β5 S759/S762 were generated by in vivo assembly^{31 (link)} with the PCR-based overlap of primers containing the point mutants using Phusion DNA polymerase (Invitrogen) and β5 Integrin-iRFP source vector.

Lukas F., Matthaeus C., López-Hernández T., Lahmann I., Schultz N., Lehmann M., Puchkov D., Pielage J., Haucke V, & Maritzen T. (2024). Canonical and non-canonical integrin-based adhesions dynamically interconvert. Nature Communications, 15, 2093.

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Zebrafish Retinal RNA Extraction and cDNA Synthesis

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Zebrafish retinas were dissected as described above and homogenized in 1 ml of TRIzol (Invitrogen, Carlsbad, CA). Total RNA was extracted following the manufacturer's protocol. Prior to cDNA synthesis, RNA was treated with DNase (Invitrogen) for 30 min at 37°C. The reaction was stopped using DNase stopping solution (Invitrogen), and the entire solution was used for cDNA synthesis. 0.5 μg of total RNA was used for cDNA synthesis using the SuperScript II Reverse Transcriptase (Invitrogen).
PCR amplification of cDNA was done using Template cDNA (20–50 ng), forward primer (1 μl [stock 10 μM]), reverse primer (1 μl [stock 10 μM]), dNTP (5 μl stock 2 mM), 5X HQ buffer (10 μl), and Phusion DNA polymerase (0.5 μl) (Invitrogen) with the following cycling conditions—95°C for 3 min followed by 35 cycles of the following temperature regime: 98°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and a final elongation at 72°C for 10 min. (See Table 2.)

Crespo C., Soroldoni D, & Knust E. (2018). A novel transgenic zebrafish line for red opsin expression in outer segments of photoreceptor cells. Developmental Dynamics, 247(7), 951-959.

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Screening for APOL1 Resistance in Trypanosomes

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The screening for APOL1 resistance was performed as in ref. 15 (link), using the library described in ref. 37 (link). The bloodstream-form RNAi library was cultivated in 10-ml flasks in HMI-9 medium containing 10% FCS and 10% Serum Plus in the presence of 1 μg ml⁻¹ geneticin and 1 μg ml⁻¹ hygromycin. For induction of RNAi, 1 μg ml⁻¹ doxycycline was added to the culture. After 1–3 days of RNAi induction, the library was diluted in the same culture medium to 5 × 10⁴ to 5 × 10⁵ cells per ml in 10-ml flasks. After treatment with 0.01 to 3% NHS, the emergence of resistant populations was monitored. Genomic DNA was extracted (Qiamp DNA minikit, Qiagen) and RNAi inserts were amplified by PCR with Phusion DNA polymerase (Invitrogen), using the p2T7-for and p2T7-rev2 primers37 (link). The PCR products were cloned into TOPO Zero blunt plasmid (Invitrogen) before bacterial transformation and sequencing. The sequences of the RNAi inserts were analysed by BLAST algorithm in NCBI, GeneDB and TriTryp databases.
The TbKIFC1 DNA fragments selected in four independent experiments were from nucleotides 1,692–2,446, 2,438–3,130 and 1,925–2,446 (two times).

Vanwalleghem G., Fontaine F., Lecordier L., Tebabi P., Klewe K., Nolan D.P., Yamaryo-Botté Y., Botté C., Kremer A., Burkard G.S., Rassow J., Roditi I., Pérez-Morga D, & Pays E. (2015). Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1. Nature Communications, 6, 8078.

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Silencing RDR1 Gene in Potato

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The silencing construct design was based on that used previously in tobacco for silencing NtRDR1 transcripts by Rakhshandehroo and colleagues36 (link) and two target sequences (Supplementary Fig. S2) were chosen by lining up known RDR1 gene sequences. DNA for the silencing constructs was amplified from potato DNA by PCR using Phusion DNA polymerase (Invitrogen, Waltham MA). Primers used for amplifying the first and second RNAi constructs are listed in Supplementary Table S2 (primer numbers 1–4). The Gateway cloning entry vector pDONR 207 (Invitrogen) was used to generate entry clones for each of the two RNAi constructs, following the manufacturers instructions. For RNAi sequences inserted into pDONR 207 plasmid, the Gateway LR reaction (Invitrogen) was used to recombine the construct sequences into the destination vector pK7GWIWG2(II)0 used for potato plant transformation62 (link).

Hunter L.J., Brockington S.F., Murphy A.M., Pate A.E., Gruden K., MacFarlane S.A., Palukaitis P, & Carr J.P. (2016). RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerases. Scientific Reports, 6, 23082.

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RNA Extraction and RNA-seq Library Preparation

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Total RNA was extracted from the tissue using TRIzol® Reagent according the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (TaKara). Then RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). Only high-quality RNA sample (OD260/280=1.8∼2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0, >1 μg) was used to construct sequencing library. RNA-seq transcriptome library was prepared using TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA), SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) and Phusion DNA polymerase (NEB) according to manufacturer's protocol. After quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the NovaSeq 6000 sequencer (2 × 150bp read length).

Yu J., Feng H., Sang Q., Li F., Chen M., Yu B., Xu Z., Pan T., Wu X., Hou J., Zhu Z., Yan C., Su L., Li J, & Liu B. (2023). VPS35 promotes cell proliferation via EGFR recycling and enhances EGFR inhibitors response in gastric cancer. eBioMedicine, 89, 104451.

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Epitope Tagging of Proteins

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The 3x FLAG epitope (DYKDHDGDYKDHDIDYKDDDDK), FLAG epitope (DYKDDDDK), or HA epitope (YPYDVPDYA) were added to the C- or N-termini of specific proteins using either a markerless mutagenesis strategy (Xie et al., 2011 (link); Zhang et al., 2017 (link)) or via allelic replacement with an antibiotic cassette. Transformation reactions were performed as follows. Bacteria were diluted 1:20 from overnight cultures and grown to an optical density of OD₆₀₀ ∼0.2 in THYE before adding 500 ng ml^–1 transforming DNA and 1 μg ml^–1 CSP. The cultures were subsequently incubated for an additional 2 h and then plated on selective media. The strains, plasmids, and primers used in this study are listed in Supplementary Tables S1, S2. Phusion DNA polymerase (NEB) was used to amplify individual PCR fragments, while AccuPrime Polymerase (Invitrogen) was used for overlap extension PCR (OE-PCR). All constructs and/or genetic modifications were verified by PCR and sequencing.

Mu R., Shinde P., Zou Z., Kreth J, & Merritt J. (2019). Examining the Protein Interactome and Subcellular Localization of RNase J2 Complexes in Streptococcus mutans. Frontiers in Microbiology, 10, 2150.

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Kanadaptin FHA Domain Cloning Protocol

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Clones were generated using Ligation Independent Cloning (LIC). The gene encoding the FHA domain of kanadaptin (UniProt: Q9BWU0 or NADAP_HUMAN, residues 149–276) was amplified by polymerase chain reaction (PCR) from the Invitrogen Ultimate collection using Phusion DNA polymerase (NEB) and forward primer, 5′-tacttccaatccatgGCCCGGGCTCCCCCC-3′ and reverse primer, 5′-tatccacctttactgttaTCCCTGCAGGATAAAGAGCCGGG-3′ (target sequence in upper case). The resulting DNA was inserted into the expression vector pNIC28-Bsa4 using LIC. The expression vector encodes an amino-terminal tobacco etch virus (TEV) protease-cleavable expression and purification tag (MHHHHHHSSGVDLGTENLYFQ/S). The DNA insert and the vector were both prepared for LIC by treatment with restriction enzyme digestion and T4 DNA polymerase. Escherichia coli MachI (Invitrogen) competent cells were transformed with the treated DNA insert and vector and dispensed on to selective LB-agar plates. The success of cloning was confirmed by DNA sequencing.

Xu Q., Deller M.C., Nielsen T.K., Grant J.C., Lesley S.A., Elsliger M.A., Deacon A.M, & Wilson I.A. (2014). Structural Insights into the Recognition of Phosphopeptide by the FHA Domain of Kanadaptin. PLoS ONE, 9(9), e107309.

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Illumina Whole-Genome Chromatin IP Sequencing

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The purified DNA products were modified for Illumina Whole-Genome Chromatin IP sequencing using an Illumina Genomic DNA Sample Prep kit as follows: size-selected DNAs were end-repaired by T4 DNA polymerase and phosphorylated by T4 DNA polymerase and T4 polynucleotide kinase. The products were incubated with Klenow DNA Polymerase (Illumina) to generate 3′ adenine overhangs and then ligated to Illumina adapters, which contain 5′ thymine overhangs. The adapter-ligated products were purified on QIAquick spin columns (Qiagen), PCR-amplified with Phusion DNA Polymerase (Finnzymes) for 30 cycles using an Illumina genomic DNA primer set. The PCR products were purified on QIAquick and MinElute columns (Qiagen).The quality of the DNA was assessed and quantified using an Agilent DNA 1000 Series II assay and NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and the DNA was diluted to 10 nM. Cluster generation and sequencing were performed using a Standard Cluster Generation kit and a Cycle Solexa Sequencing kit on the Illumina Cluster Station and Illumina HiSeq2000 sequencer following the manufacturer's instructions. Sequencing was carried out by the Research & Cooperation Division, BGI-Shenzhen.

Liu H., Zhou P., Lan H., Chen J, & Zhang Y.X. (2017). Comparative analysis of Notch1 and Notch2 binding sites in the genome of BxPC3 pancreatic cancer cells. Journal of Cancer, 8(1), 65-73.

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