Phusion hs 2 dna polymerase

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Phusion HS II DNA polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It provides enhanced speed and robustness for a wide range of PCR applications.

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Lab products found in correlation

Gibson assembly master mix, by New England Biolabs (2 mentions) Qiaprep miniprep kit, by Qiagen (2 mentions) Snapgene software, by GSL Biotech (2 mentions) Plasmid midiprep kit, by Qiagen (1 mentions) Phusion hf buffer, by Thermo Fisher Scientific (1 mentions) Dneasy plant mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

Phusion HS II polymerase (10 citations) Phusion DNA polymerase (487 citations) Phusion HS II (8 citations) Phusion polymerase (407 citations) DNA polymerase (71 citations)

5 protocols using phusion hs 2 dna polymerase

Emulsion PCR for Library Amplification

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The selected library was amplified by emulsion PCR in order to prevent template switching. The oil phase, made fresh and chilled on ice before addition to the aqueous phase, was composed of 92.95% Mineral Oil, 7% ABIL WE-09 and 0.05% Triton X-100, then thoroughly vortexed. The PCR mixture, or aqueous phase, was prepared in a 1.5 ml screw cap tube as follows: 0.5 μg/μl BSA, 0.2 mM dNTPs mix, 0.4 μM of each primer (P5/P7 PCR) or 5 μl of each primer (Nextera PCR), 0.5 μl Phusion HS II DNA polymerase (Thermo Scientific, Waltham, USA) and 1X Phusion Green buffer, together with either 1.5 × 10⁹ or 7.5 × 10⁹ plasmid copies (depending on the number of PCR cycles). dH₂O was added to a final volume of 50 ul.
The water-in-oil emulsion was created by addition of 9 volumes of the oil phase on top of the aqueous phase and vigorous shaking of the tube in an MP FastPrep-24 Tissue and Cell Homogenizer (Hyland Scientific), for 5 min at a speed of 4 m/s. After shaking, the emulsified product was divided into six PCR tubes (50 μl to each) that were overlaid with 10 μl of mineral oil each to prevent the emulsion from breaking during the PCR program, due to extreme heating. Non-emulsified control reactions were utilized as controls.

Davidsson M., Diaz-Fernandez P., Schwich O.D., Torroba M., Wang G, & Björklund T. (2016). A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing. Scientific Reports, 6, 37563.

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Gibson Assembly and Plasmid Preparation

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Gibson assembly primers were designed using Snapgene software (GSL Biotech) and sequencing primers were identified using the GenScript sequencing primer tool. All primers were synthesized by IDT. Primer sequence and a brief description of their use are provided in Supplementary Data 15. Polymerase chain reaction products were amplified using Phusion HS II DNA polymerase (F549; Thermo Fisher). Gibson Assembly was conducted using Gibson Assembly Master Mix (E2611; NEB) according to the recommended protocol. Plasmids were transformed into NEB5α cells (C2987; NEB), prepared using the QIAprep Miniprep Kit (27104; Qiagen) or the Qiagen Plasmid Midiprep Kit (12143; Qiagen), and sequence verified using the Sanger method at the Johns Hopkins School of Medicine Synthesis & Sequencing Facility.

Keener R., Chhetri S.B., Connelly C.J., Taub M.A., Conomos M.P., Weinstock J., Ni B., Strober B., Aslibekyan S., Auer P.L., Barwick L., Becker L.C., Blangero J., Bleecker E.R., Brody J.A., Cade B.E., Celedon J.C., Chang Y.C., Cupples L.A., Custer B., Freedman B.I., Gladwin M.T., Heckbert S.R., Hou L., Irvin M.R., Isasi C.R., Johnsen J.M., Kenny E.E., Kooperberg C., Minster R.L., Naseri T., Viali S., Nekhai S., Pankratz N., Peyser P.A., Taylor K.D., Telen M.J., Wu B., Yanek L.R., Yang I.V., Albert C., Arnett D.K., Ashley-Koch A.E., Barnes K.C., Bis J.C., Blackwell T.W., Boerwinkle E., Burchard E.G., Carson A.P., Chen Z., Chen Y.D., Darbar D., de Andrade M., Ellinor P.T., Fornage M., Gelb B.D., Gilliland F.D., He J., Islam T., Kaab S., Kardia S.L., Kelly S., Konkle B.A., Kumar R., Loos R.J., Martinez F.D., McGarvey S.T., Meyers D.A., Mitchell B.D., Montgomery C.G., North K.E., Palmer N.D., Peralta J.M., Raby B.A., Redline S., Rich S.S., Roden D., Rotter J.I., Ruczinski I., Schwartz D., Sciurba F., Shoemaker M.B., Silverman E.K., Sinner M.F., Smith N.L., Smith A.V., Tiwari H.K., Vasan R.S., Weiss S.T., Williams L.K., Zhang Y., Ziv E., Raffield L.M., Reiner A.P., Arvanitis M., Greider C.W., Mathias R.A, & Battle A. (2024). Validation of human telomere length multi-ancestry meta-analysis association signals identifies POP5 and KBTBD6 as human telomere length regulation genes. Nature Communications, 15, 4417.

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Genotyping Grsf1 Knockout Mice

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Mouse tail or ear biopsies were incubated in 1.5 ml tubes for 30 min in 40 µl lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 12) at 95 °C under mild agitation. Precipitate was spun down at 15,000 rpm for 30 s, 40 µl of neutralizing buffer (40 mM Tris/HCl pH 5.0) were added, the liquid was recovered and the sample was stored on ice until further use or was stored at − 20 °C. The following primer combinations were used for genotyping. Wildtype mice: Grsf1 forward, 5ʹ-TCA GTG AAG AAC GCT CTT GTT GGC-3ʹ and 24140 down: 5ʹ-GAA CTT CTT GGA TTC TGG CTC ACA-3ʹ (556 bp amplification product). Grsf1^−/− mice: Grsf1 5ʹarm 5ʹ-TTT GTG TGG TAG GGT TCA CGT GGG-3ʹ and 24140 down 5ʹ-GAA CTT CTT GGA TTC TGG CTC ACA-3ʹ (682 bp amplification product). The PCR reaction mixture containing 2 µl genomic DNA, 0.4 µl dNTPs (10 mM each), 1 µl Primer Grsf1 (5 µM), 2 µl Primer 24140do (5 µM), 1 µl Primer Grsf1 5'arm (5 µM), 4 µl Phusion HF Buffer, 0.6 µl DMSO, 0.2 µl Phusion HS II DNA Polymerase (Thermofisher Scientific, Dreieich, Germany) and 8.8 µl sterile water was incubated according to the following PCR protocol: initial denaturation 1 min 98 °C; 31 × cycle: 20 s at 98 ℃ denaturation, 30 s 68 ℃ annealing, 30 s 72 ℃ extension; final extension 3 min 72 ℃. PCR products were separated in an 1.5% agarose gel and were visualized on an UV-gel imager.

Dumoulin B., Heydeck D., Jähn D., Lassé M., Sofi S., Ufer C, & Kuhn H. (2022). Male guanine-rich RNA sequence binding factor 1 knockout mice (Grsf1−/−) gain less body weight during adolescence and adulthood. Cell & Bioscience, 12, 199.

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Efficient DNA Cloning Strategies

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Plasmids were constructed using PCR fragments and restriction cloning or Gibson assembly methods using Gibson Assembly Master Mix (New England Biolabs E5510). Plasmids and assembly templates were designed in silico using SnapGene software (GSL Biotech) and then constructed using PCR with Phusion HS II DNA polymerase (Thermo Fisher F549) from genomic DNA, synthetic G-blocks (IDT), and/or plasmids, followed by TA-cloning and/or Gibson assembly. Plasmids were transformed into NEB5α competent cells (NEB C2987H) for Gibson Assembly or TOP10 competent cells (Thermo Fisher K204040) for TA cloning. Plasmids were prepared using QIAprep Miniprep Kit (Qiagen 27106). Constructs were confirmed by restriction digest and Sanger sequencing. Constructs used to generate strains, including the plasmid or genomic DNA template and oligonucleotides or enzymes used to prepare the homologous repair DNA, are listed in Supplementary file 1. All oligonucleotides and synthetic G-blocks were generated from IDT.

Shubin C.B, & Greider C.W. (2020). The role of Rif1 in telomere length regulation is separable from its role in origin firing. eLife, 9, e58066.

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PCR Amplification of Fungal Gene Clusters

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Genomic DNA was extracted from fresh mycelium using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Table 1-2) for PCR amplification of gene clusters were designed using the Primer3Plus software (Untergasser et al., 2007) (link) based on available Fp genomic sequence (Gardiner et al., 2012) (link). The parameters were set to identify primers producing 3-4 kb products with at least 50 bp of homology to neighboring PCR product or the shuttle vector. Genes included in constructs were based on previous gene cluster characterization; FCK (Sørensen et al., 2018) (link), W493 (J. L. Sørensen et al., 2014) . Full gene clusters were PCR-amplified in 3168-3941 bp segments with at least 59 bp neighboring overlap using the Phusion HS II DNA polymerase (Thermo Fisher scientific) following manufacturer's instructions. For amplification of the FCK cluster, the two outermost primers had a 36 bp tail homologous to shuttle vector's multiple cloning site.

Nielsen M.R., Wollenberg R.D., Westphal K.R., Sondergaard T.E., Wimmer R., Gardiner D.M, & Sørensen J.L. (2019). Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum. Fungal genetics and biology : FG & B, 132.

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