Phusion hf buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, Finland

Phusion HF buffer is a high-fidelity buffer designed for use with the Phusion DNA polymerase. It is formulated to provide optimal performance and accuracy during PCR amplification.

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50 protocols using phusion hf buffer

Amplification of MPRA Libraries

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Round #1 PCR mixtures (50 μl) contained 10 pg of MPRA library-71 or library-83 as template, 1 × Phusion HF Buffer (Thermo Fisher Scientific), 200 μM dNTPs, 0.5 μM primers Libr-A_N-for/Libr-rev (Table 3), 0.5 mg/ml BSA (EUR_X) and 2 U of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Amplification was performed under the following conditions: 95 °C for 1 min; 15 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min. The PCR products were column-purified with the GeneJET PCR Purification Kit (Thermo Fisher Scientific) and eluted in 50 μl of nuclease-free water.
Round #2 PCR mixtures (50 μl) contained 0.5 μl of the purified round #1 PCR products as template, 1 × Phusion HF Buffer (Thermo Fisher Scientific), 200 μM dNTPs, 0.5 μM primers Libr-P5-for/Libr-P7-rev (Table 3), 0.5 mg/ml BSA (EUR_X) and 2 U of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Amplification was performed under the following conditions: 95 °C for 1 min; 18 cycles of 95 °C for 10 s, 52 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min.

Omelina E.S., Ivankin A.V., Letiagina A.E, & Pindyurin A.V. (2019). Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries. BMC Genomics, 20(Suppl 7), 536.

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Optimized ePCR Amplification Protocol

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Round #1 ePCR mixtures (50 μl) contained 10 pg, 100 pg or 1 ng of equimolar mixture of plasmid#1 and plasmid#2 or 10 pg of MPRA library-71 or library-83 as template, 1 × Phusion HF Buffer (Thermo Fisher Scientific), 200 μM dNTPs, 0.5 μM primers Libr-A_N-for/Libr-rev (Table 3), 0.5 mg/ml BSA (EUR_X) and 2 U of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Amplification was performed under the following conditions: 95 °C for 1 min; 15 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 10 or 30 s (for details, see Results); 72 °C for 5 min. Aliquots of 10 μl of the purified reaction products were analyzed by agarose gel electrophoresis.
Round #2 ePCR mixtures (50 μl) contained 0.3 or 0.5 μl of the purified round #1 ePCR products as template (for details, see Results), 1 × Phusion HF Buffer (Thermo Fisher Scientific), 200 μM dNTPs, 0.5 μM primers Libr-P5-for/Libr-P7-rev (Table 3), 0.5 mg/ml BSA (EUR_X) and 2 U of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). Amplification was performed under the following conditions: 95 °C for 1 min; 18 or 20 cycles (for details, see Results) of 95 °C for 10 s, 52 °C for 30 s, and 72 °C for 10 or 30 s (for details, see Results); 72 °C for 5 min. Aliquots of 10 μl of the purified reaction products were analyzed by agarose gel electrophoresis.

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Quantifying DNA Damage in Tissues

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Overall DNA damage (fragmentation and base modifications) in skin and muscle samples was assayed by PCR amplification using Phusion polymerase (Thermo Scientific), a highly accurate and thermostable recombinant DNA polymerase that stalls at most miscoding lesions, such as cytosine deaminations and abasic sites [41] (link). Four overlapping primers sets in both the nuclear and mitochondrial genomes targeting sequences of increasing length from 1000–8000 bp were PCR-amplified (Tables 2 and 3) for all sampled days post-mortem. The targets, which span the mitochondrial HVRI and the autosomal STR marker D18S51, were chosen to provide identity information that could be used to detect and exclude possible contamination. Each 20 µL reaction contained 1× Phusion HF buffer (Finnzymes), 0.5 µM forward and 0.5 µM reverse primers (Microsynth), 0.2 mM each dNTP, 0.25 µL Phusion DNA polymerase and 2 µL DNA extract (5 ng/µL). The PCR cycling conditions were as follows: 98°C for 30 s, 30 cycles of 98°C for 10 s, 66–68°C for 20 s, and 72°C for 30–240 s (product length-dependent), and a final extension at 72°C for 10 min. All amplified products were first visualized by agarose gel electrophoresis and then sequenced and compared to the LL day 0 consensus sequences in order to exclude potential non-endogenous amplifications.

Shved N., Haas C., Papageorgopoulou C., Akguel G., Paulsen K., Bouwman A., Warinner C, & Rühli F. (2014). Post Mortem DNA Degradation of Human Tissue Experimentally Mummified in Salt. PLoS ONE, 9(10), e110753.

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Isolation and PCR Amplification of DNA

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Total DNA was isolated with FlexiGene DNA kit (Qiagen) from BJ (passage 4), HFF1 (passage 4), BJ-derived iB4 and iB5 (both passage 18), and HFF1-derived iPS2 and iPS4 (both passage 18). 50 ng of DNA was used as PCR template. PCR reactions contained 1x Phusion HF Buffer, 3% DMSO and 0.6 Units of proof-reading Phusion Hot Start II High Fidelity Polymerase (all from Finnzymes, Espoo, Finland). The reactions were carried out using Dyad Thermal Cycler (BioRad, Hercules, CA, USA) according to the following program: 98°C for 30s, followed by 30 cycles of 98°C for 10s / 58°C for 30s / 72°C for 30s, and a final extension step at 72°C for 5min. Primers sequences are reported in Supp. Table 1.

Prigione A., Lichtner B., Kuhl H., Struys E.A., Wamelink M., Lehrach H., Ralser M., Timmermann B, & Adjaye J. (2014). Human iPSCs harbor homoplasmic and heteroplasmic mitochondrial DNA mutations while maintaining hESC-like metabolic reprogramming. Stem cells (Dayton, Ohio), 32(2), 364-376.

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16S rRNA Gene Amplification Protocol

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PCR reactions were performed in 50 μl volumes containing: 0.2 mM each primer V1-63f 5’-GCCTAACACATGCAAGTC-3' [23 (link)] and 518r 5’-ATTACCGCGGCTGCTGG-3' [24 (link)], 0.2 mM each dNTP, 1 x Phusion HF buffer (Finnzymes), 0.5 units Phusion™ High-Fidelity DNA Polymerase (Finnzymes), 10 ng of pooled canine plaque DNA and ddH₂O. PCR cycling conditions were as follows; 98°C for 45 s, 20 cycles of 98°C for 10 s, 55°C for 30 s and 72°C for 15 s, and a final extension of 72°C for 8 min. To minimise PCR bias, 20 cycles of amplification were performed in 8 separate replicate assays, and the PCR reactions were subsequently pooled. PCR amplification products were visualised using 1% agarose gel electrophoresis and fragments of the expected size (~460 bp) were excised from the agarose gel and purified using a QiaQuick Gel Extraction kit (Qiagen) following the manufacturer’s protocol. Gel extracted and purified V1-V3 16S rRNA gene amplification products were subsequently pooled and quantified using a Qubit™ fluorimeter (Invitrogen) and stored at -20°C prior to library preparation for 454 pyrosequencing (Accession no: SRR830918).

McDonald J.E., Larsen N., Pennington A., Connolly J., Wallis C., Rooks D.J., Hall N., McCarthy A.J, & Allison H.E. (2016). Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules. PLoS ONE, 11(6), e0157046.

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Genetic Analysis of Exons in NDP, FZD4, LRP5, and TSPAN12

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Entire NDP, FZD4, LRP5, and TSPAN12 exons were amplified using sets of primers from previous reports.[1 (link)12 (link)23 (link)] Polymerase chain reaction (PCR) was performed in a 20 μl reaction volume using the 2 U Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), 200 μM of each dNTP, 10 μM of each primer, 70 ng of DNA template, and 4 μl of 5 × Phusion HF buffer (Thermo Fisher Scientific, Waltham, MA, USA).
Initial denaturation was performed at 98°C for 30 s, followed by 30 cycles of denaturation, annealing at respective temperatures (available in references) and extension at 72°C. Final extension time was five minutes. Gel electrophoresis using a 3% agarose gel was performed to analyze amplified fragments, the sizes of which were determined using 50 bp DNA marker. Favorable PCR products were purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany) and sequenced.

Mohd Khair S.Z., Ismail A.S., Embong Z, & Mohamed Yusoff A.A. (2019). Detection of FZD4, LRP5 and TSPAN12 Genes Variants in Malay Premature Babies with Retinopathy of Prematurity. Journal of Ophthalmic & Vision Research, 14(2), 171-178.

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Amplification and sequencing of cDNA targets

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For a 20 µl reaction, the following reagents were included: 4 µl of 5× Phusion HF Buffer (Thermo Fisher Scientific), 0.4 µl of 10 mM dNTP, 1 µl of 40 µM sense primer (Integrated DNA Technologies), 1 µl of 40 µM antisense primer (Integrated DNA Technologies), 0.2 µl of Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), 1 µl of 1/3 diluted cDNA template, and sterile H₂O.
PCR was performed in a thermal cycler. The initial denaturation was 98 °C for 40 s followed by a cycle of denaturation at 98 °C for 20 s, annealing at 62 °C for 25 s and extension at 72 °C for 4 min. The cycle was repeated 30 times. A final extension was at 72 °C for 10 min. The PCR products were separated on a 1% agarose gel for approximately 1 h at 110 V and visualized with ethidium bromide.
The PCR products were gel extracted, and the concentration of DNA was measured according to the same procedure followed for products derived from genomic DNA (see above). Samples were sequenced using specific primers covering the entire range of PCR products (Table 2). Sequence chromatogram files where edited and aligned with NCBI reference sequence NM_000390.2 by ChromasPro (Technelysium).

Furgoch M.J., Mewes-Arès J., Radziwon A, & MacDonald I.M. (2014). Molecular genetic diagnostic techniques in choroideremia. Molecular Vision, 20, 535-544.

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Bacterial 16S rRNA Gene Sequencing of BAL

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Cell-free BAL specimens were stored at −80 °C until analyzed in batch. Microbial DNA was first extracted using the QIAGEN DNeasy PowerMax Soil kit following the manufacturer’s instructions. Then, PCR amplification was performed targeting the bacterial 16S rRNA gene V5-V6 region using primers 799F and 1115R [17 (link)]. We conducted PCR in a triplicate 25 μL mixture containing 5 μL 5× Phusion HF Buffer (Thermo Fisher Scientific, Frederick, MD, USA), 0.5 μL dNTPS (10 μmol/L each), 0.75 μL DMSO, 0.5 μL each primer (10 μmol/L), 0.25 μL Phusion Hot Start II polymerase (2 U/μL) (Thermo Fisher Scientific), 1 μL DNA template and 16.5 μL molecular-grade H₂O. PCR reactions were performed using the following condition: 30 s initial denaturation at 98 °C, followed by 35 cycles of 15 s at 98 °C, 30 s at 64 °C, and 30 s at 72 °C, with a final 10 min elongation at 72 °C. PCR products were first normalized using a SequalPrep Normalization kit (Thermo Fisher Scientific), then pooled and purified using AMPure (Beckman Coulter Life Sciences, Brea, CA, USA) to avoid contaminants. After that, we prepared the DNA library by mixing equimolar concentrations of DNA for each sample and sequenced the DNA using Illumina MiSeq reagent kit v3 (Illumina, Hayward, CA, USA) [8 (link)].

Wang Z., Jenabian M.A., Alexandrova Y., Pagliuzza A., Olivenstein R., Samarani S., Chomont N., Kembel S.W, & Costiniuk C.T. (2022). Interplay between the Lung Microbiome, Pulmonary Immunity and Viral Reservoirs in People Living with HIV under Antiretroviral Therapy. Viruses, 14(11), 2395.

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Genotyping Mice from Tail Biopsies

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Genotype was determined from tail biopsies on 10-day old pups. Crude genomic DNA was extracted by DirectPCR Lysis Reagent-Tail (Viagen Biotech, Cat # 101-T) according to the manufacturer’s instructions. Phusion Hot Start II High-Fidelity DNA polymerase kit (Thermo Scientific) was used. The PCR reaction contained 5 μl of diluted genomic DNA, 4 μl of 5 × Phusion HF Buffer, 0.4 μl of dNTP mix (dATP, dCTP, dGTP, and dTTP at 10 mM, Thermo Scientific), 2 μl of each primer at 0.5 nM, and 0.2 μl of Phusion polymerase and H₂O in a total volume of 20 ml. PCRs were done under the following conditions: 98°C for 30 s followed by 30 cycles of 98°C for 8 s, 62°C for 30 s, and 72°C for 1 min and a final elongation step for 5 min at 72°C in a T100 thermocycler (Bio-Rad). Ten microliters of PCR product were then digested in a final volume of 20 μl added to 0.2 μl of BspHI enzyme and 2 μl of enzyme buffer. PCR outcome was analyzed on a 2% agarose gel. Information about primer sequences is shown in Supplementary Table 2.

Xue Y., Kremer M., Muniz Moreno M.D., Chidiac C., Lorentz R., Birling M.C., Barrot M., Herault Y, & Gaveriaux-Ruff C. (2022). The Human SCN9AR185H Point Mutation Induces Pain Hypersensitivity and Spontaneous Pain in Mice. Frontiers in Molecular Neuroscience, 15, 913990.

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Mitochondrial D-loop Region Amplification

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Three sets of primers were designed to amplify or target three independent but overlapping fragments that span the whole D-loop region, as listed in Table I. Briefly, PCR was performed on the SureCycler 8800 Thermal Cycler (Agilent Technologies, Inc., Santa Clara, CA, USA), in a 50 µl reaction mixture containing 100 ng DNA template, 200 µM each dNTP, 20 pMol each primer, 10 µl 5X Phusion HF buffer (Thermo Fisher Scientific, Inc.) and 2 U Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc.).
The cycling profile was set at 98°C initial denaturation for 1 min, followed by 35 cycles of denaturation at 98°C for 20 sec, annealing at 56°C for 20 sec, extension at 72°C for 20 sec and a final extension at 72°C for 5 min. The amplified PCR fragments were analyzed via 2% agarose gel electrophoresis using a GeneRuler 100 bp DNA ladder (Thermo Fisher Scientific, Inc.) in order to determine the expected size of the amplified PCR fragments. The PCR products with the expected size were purified with the QIAquick PCR Purification kit (Qiagen GmbH) according to the manufacturer's protocol and stored at −20°C until use for DNA sequencing analysis.

Mohamed Yusoff A.A., Mohd Nasir K.N., Haris K., Mohd Khair S.Z., Abdul Ghani A.R., Idris Z, & Abdullah J.M. (2017). Detection of somatic mutations in the mitochondrial DNA control region D-loop in brain tumors: The first report in Malaysian patients. Oncology Letters, 14(5), 5179-5188.

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