phusion dna polymerase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher phusion dna polymerase
    Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    phusion dna polymerase - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins
    Article Snippet: Paragraph title: 2.4. PCR Amplification of Control and Hypothetical Proteins of Unknown Function Genes for Cloning ... For each reaction, 0.5 μM of primers, 0.2 mM of dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA), 10 or 6 μL of 5× Phusion Buffer and 0.02 U/μL Phusion DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) were added.

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: Paragraph title: Cloning and expression of GroEL. ... PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture.

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: To create PVX expressing 6×‐2b‐HA, the tagged 2b protein was amplified by PCR with appropriate oligos and cloned into Cla I/ Sal I‐linearized construct pgR 107. .. All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: Candidate resistance genes were then synthesised (GeneArt, ThermoScientific, USA) and cloned into the pUASTattB plasmid (GenBank: EF362409.1 ). .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in .

    Article Title: Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins
    Article Snippet: Complementation assays Klebsiella genome regions, containing ExbB , ExbBD , OmpC , FhuA , TonB and FimB gene ORFs along with 5′ non-coding promoter regions, were PCR-amplified from K . quasipneumoniae DSM 28212 genomic DNA with help of Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated in pJET1.2 (Thermofisher Scientific Baltics). .. After sequencing, cloned fragments were excised with a pair of restriction endonucleases specific for each fragment, ligated in pACYC184 (NEB) and transformed into the respective K . quasipneumoniae mutants.

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: .. 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). ..

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: Paragraph title: General methods and cloning. ... All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher).

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). .. The genes for Ml -Fd and sFd-35-ER were cloned into pET-28b to create expression vectors for protein purification.

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: To generate the CRISPR/Cas9 construct targeting S2 -SLF1, the pre-tRNA and the gRNA scaffold fragments were ligated to the S2 -SLF1 - Protospacer-9 ( S2 -SLF1 -PS9) fragment, and the resulting DNA fragment was cloned into pKSE401 plasmid ( ; a gift from Qi-Jun Chen; Addgene plasmid # 62202) following the protocol described previously ( ; ). .. The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific).

    Amplification:

    Article Title: A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins
    Article Snippet: Paragraph title: 2.4. PCR Amplification of Control and Hypothetical Proteins of Unknown Function Genes for Cloning ... For each reaction, 0.5 μM of primers, 0.2 mM of dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA), 10 or 6 μL of 5× Phusion Buffer and 0.02 U/μL Phusion DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) were added.

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: .. PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture. .. The purified 1.65-kb reaction product was digested with the NdeI and EcoRI restriction enzymes and inserted into a pET28 cloning vector previously digested with the same enzymes.

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: .. Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: To create PVX expressing 6×‐2b‐HA, the tagged 2b protein was amplified by PCR with appropriate oligos and cloned into Cla I/ Sal I‐linearized construct pgR 107. .. All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: Genotyping of the OATP1B1 c. 521 T > C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol
    Article Snippet: In a 20 μl reaction volume containing dNTPs (0.5 mM each), the primer pair (0.5 μM each), 1x Phusion buffer, and 2 units of Phusion DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), PCR efficiency was compared among reactions containing different amounts of template DNA of 10, 50, 100 or 200 ng. .. As shown in , for the liver FFPE tissues (#4 and #5), 50 and 100 ng template DNA per 20 μl reaction yielded a much higher amount of amplification of the expected 339 bp band compared to 10 ng template DNA.

    Article Title: Role of rhesus macaque IFITM3(2) in simian immunodeficiency virus infection of macaques
    Article Snippet: However, sequencing results of mamuIFITM3(2)gen5-1for/MamuIFITM3(2)gen3 showed no difference to the mamuIFITM3gen5-1forA/MamuIFITM3(2)gen3 amplification, indicating high specificity. .. Reaction mixtures contained 100 ng genomic DNA, 50 pmol of each primer, 100 μM dNTP mix, 10% betain, 1x HF synthesis buffer and 1 U Phusion DNA polymerase (ThermoFisher).

    Article Title: Survival of Polyploid hybrid salamander embryos
    Article Snippet: .. Specific PCR conditions For the lab-reared egg samples, we amplified the D-loop from 1 μl of extracted egg DNA using Phusion DNA polymerase (Thermo Scientific) and 2 μl of mixed 10 μM DL1/007 primer pair [ ] in 25 μl total reaction volumes. .. We cycled each reaction on a Perkin Elmer 9600 thermocycler as follows: initial denaturation at 98 °C for 120 s, followed by 35 cycles of 98 °C:10 s; 55 °C:15 s; and 72 °C:30 s. We confirmed clean amplification by agarose gel electrophoresis and purified the PCR products by Promega (Madison, Wisconsin) Wizard SV Gel and PCR Cleanup System according to manufacturer’s protocol.

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: .. 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). ..

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Article Title: Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea
    Article Snippet: Paragraph title: 16s rRNA amplification and sequencing ... Polymerase chain reactions were performed using Phusion DNA polymerase (Thermo Scientific, Espoo, Finland) on a MyCycler thermocycler for 25 cycles of denaturation at 98 °C for 10 s, annealing at 69 °C for 30 s, and extension at 72 °C for 30 s. The amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Hilden, Germany) and were quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: .. These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). ..

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific). .. The assembled product was further amplified using S51AD5-F and S3AD5-R primers, and it was digested with Fok I (New England BioLabs).

    Synthesized:

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). .. The genes encoding Mastigocladus laminosus , Zea mays , Chlamydomonas reinhardtii , and cyanomyophage PSSM-2 Fd were synthesized as G-blocks by Integrated DNA Technologies.

    Construct:

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. All constructs were verified by Sanger sequencing.

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: Paragraph title: Binary vector constructs ... All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: Using the PhiC31 system, constructs were transformed into the germline of a D. melanogaster strain carrying an attP docking site on chromosome 2 (attP40) and the phiC31 integrase gene under the control of the vasa regulatory region on the X chromosome [y w M(eGFP, vas-int, dmRFP)ZH-2A; P{CaryP}attP40] ( ). .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in .

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: .. These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). ..

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: To generate the CRISPR/Cas9 construct targeting S2 -SLF1, the pre-tRNA and the gRNA scaffold fragments were ligated to the S2 -SLF1 - Protospacer-9 ( S2 -SLF1 -PS9) fragment, and the resulting DNA fragment was cloned into pKSE401 plasmid ( ; a gift from Qi-Jun Chen; Addgene plasmid # 62202) following the protocol described previously ( ; ). .. The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific).

    Incubation:

    Article Title: Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance
    Article Snippet: Briefly, for each sample, a mixture including the total RNA and 5 pmol of oligonucleotide AC‐203 or AC‐207 (for TSs in segments L and M, respectively) (Table ) was incubated for 1.5 min at 98°C, and then transferred to ice. .. PCR to amplify TS fragments from segment L or segment M (604 bp or 537 bp, respectively) was performed using oligonucleotide pairs AC‐205/AC‐206 or AC‐209/AC‐210 (Table ), respectively, and Phusion DNA polymerase (ThermoFisher Scientific) with the following program: 30 s at 98°C; 32 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; 10 min at 72°C.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Genotyping of the OATP1B1 c. 521 T > C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol
    Article Snippet: We optimized the amount of the extracted FFPE DNA used as template in PCR. .. In a 20 μl reaction volume containing dNTPs (0.5 mM each), the primer pair (0.5 μM each), 1x Phusion buffer, and 2 units of Phusion DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), PCR efficiency was compared among reactions containing different amounts of template DNA of 10, 50, 100 or 200 ng.

    Spectrophotometry:

    Article Title: Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea
    Article Snippet: .. Polymerase chain reactions were performed using Phusion DNA polymerase (Thermo Scientific, Espoo, Finland) on a MyCycler thermocycler for 25 cycles of denaturation at 98 °C for 10 s, annealing at 69 °C for 30 s, and extension at 72 °C for 30 s. The amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Hilden, Germany) and were quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Three independent PCR amplicons obtained from the individual coral metagenomic sample were performed.

    Expressing:

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: Paragraph title: Cloning and expression of GroEL. ... PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture.

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: To create PVX expressing 6×‐2b‐HA, the tagged 2b protein was amplified by PCR with appropriate oligos and cloned into Cla I/ Sal I‐linearized construct pgR 107. .. All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: Paragraph title: Transgenic expression of candidate genes in D. melanogaster ... The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in .

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: .. 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). ..

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). .. The genes for Ml -Fd and sFd-35-ER were cloned into pET-28b to create expression vectors for protein purification.

    Transformation Assay:

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture. .. For the expression of GroEL in E. coli cells, plasmid DNA from a positive clone was transformed into BL21 cells for protein expression, and the protein obtained after induction with IPTG was purified using a Ni-agarose column as described above.

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: Using the PhiC31 system, constructs were transformed into the germline of a D. melanogaster strain carrying an attP docking site on chromosome 2 (attP40) and the phiC31 integrase gene under the control of the vasa regulatory region on the X chromosome [y w M(eGFP, vas-int, dmRFP)ZH-2A; P{CaryP}attP40] ( ). .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in .

    Article Title: Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins
    Article Snippet: Complementation assays Klebsiella genome regions, containing ExbB , ExbBD , OmpC , FhuA , TonB and FimB gene ORFs along with 5′ non-coding promoter regions, were PCR-amplified from K . quasipneumoniae DSM 28212 genomic DNA with help of Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated in pJET1.2 (Thermofisher Scientific Baltics). .. After sequencing, cloned fragments were excised with a pair of restriction endonucleases specific for each fragment, ligated in pACYC184 (NEB) and transformed into the respective K . quasipneumoniae mutants.

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). .. M. smegmatis mc2 155 cells were transformed with the pLNA29 plasmid and the transformant colonies were verified by colony PCR.

    Inhibition:

    Article Title: Genotyping of the OATP1B1 c. 521 T > C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol
    Article Snippet: “The more the better” is not better due to possible inhibition of the PCR reaction by excess DNA. .. In a 20 μl reaction volume containing dNTPs (0.5 mM each), the primer pair (0.5 μM each), 1x Phusion buffer, and 2 units of Phusion DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), PCR efficiency was compared among reactions containing different amounts of template DNA of 10, 50, 100 or 200 ng.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance
    Article Snippet: Sequence analysis of target sites in TSWV RNAs The TS sequences from viral progeny of TSWV‐infected transgenic plants were analyzed by RT‐PCR followed by Sanger sequencing. cDNA was obtained from 1–5 μg of total RNA from apical S. lycopersicum leaves using RevertAid reverse transcriptase (RT; ThermoFisher Scientific). .. PCR to amplify TS fragments from segment L or segment M (604 bp or 537 bp, respectively) was performed using oligonucleotide pairs AC‐205/AC‐206 or AC‐209/AC‐210 (Table ), respectively, and Phusion DNA polymerase (ThermoFisher Scientific) with the following program: 30 s at 98°C; 32 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; 10 min at 72°C.

    Generated:

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Polymerase Chain Reaction:

    Article Title: A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins
    Article Snippet: Paragraph title: 2.4. PCR Amplification of Control and Hypothetical Proteins of Unknown Function Genes for Cloning ... For each reaction, 0.5 μM of primers, 0.2 mM of dNTP mix (Thermo Fisher Scientific, Waltham, MA, USA), 10 or 6 μL of 5× Phusion Buffer and 0.02 U/μL Phusion DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) were added.

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: .. PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture. .. The purified 1.65-kb reaction product was digested with the NdeI and EcoRI restriction enzymes and inserted into a pET28 cloning vector previously digested with the same enzymes.

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: To create PVX expressing 6×‐2b‐HA, the tagged 2b protein was amplified by PCR with appropriate oligos and cloned into Cla I/ Sal I‐linearized construct pgR 107. .. All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: Genotyping of the OATP1B1 c. 521 T > C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol
    Article Snippet: .. In a 20 μl reaction volume containing dNTPs (0.5 mM each), the primer pair (0.5 μM each), 1x Phusion buffer, and 2 units of Phusion DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), PCR efficiency was compared among reactions containing different amounts of template DNA of 10, 50, 100 or 200 ng. .. As shown in , for the liver FFPE tissues (#4 and #5), 50 and 100 ng template DNA per 20 μl reaction yielded a much higher amount of amplification of the expected 339 bp band compared to 10 ng template DNA.

    Article Title: Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance
    Article Snippet: .. PCR to amplify TS fragments from segment L or segment M (604 bp or 537 bp, respectively) was performed using oligonucleotide pairs AC‐205/AC‐206 or AC‐209/AC‐210 (Table ), respectively, and Phusion DNA polymerase (ThermoFisher Scientific) with the following program: 30 s at 98°C; 32 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; 10 min at 72°C. ..

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in . ..

    Article Title: Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins
    Article Snippet: .. Complementation assays Klebsiella genome regions, containing ExbB , ExbBD , OmpC , FhuA , TonB and FimB gene ORFs along with 5′ non-coding promoter regions, were PCR-amplified from K . quasipneumoniae DSM 28212 genomic DNA with help of Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated in pJET1.2 (Thermofisher Scientific Baltics). .. After sequencing, cloned fragments were excised with a pair of restriction endonucleases specific for each fragment, ligated in pACYC184 (NEB) and transformed into the respective K . quasipneumoniae mutants.

    Article Title: Role of rhesus macaque IFITM3(2) in simian immunodeficiency virus infection of macaques
    Article Snippet: Reaction mixtures contained 100 ng genomic DNA, 50 pmol of each primer, 100 μM dNTP mix, 10% betain, 1x HF synthesis buffer and 1 U Phusion DNA polymerase (ThermoFisher). .. PCR cycling started with 5 min denaturation followed by 40 cycles of denaturation (30 s at 95°C), annealing (30 s at 68°C) and elongation (1 min 40 s at 72°C) and concluded with 10 min elongation at 72°C and cooling to 12°C.

    Article Title: Survival of Polyploid hybrid salamander embryos
    Article Snippet: .. Specific PCR conditions For the lab-reared egg samples, we amplified the D-loop from 1 μl of extracted egg DNA using Phusion DNA polymerase (Thermo Scientific) and 2 μl of mixed 10 μM DL1/007 primer pair [ ] in 25 μl total reaction volumes. .. We cycled each reaction on a Perkin Elmer 9600 thermocycler as follows: initial denaturation at 98 °C for 120 s, followed by 35 cycles of 98 °C:10 s; 55 °C:15 s; and 72 °C:30 s. We confirmed clean amplification by agarose gel electrophoresis and purified the PCR products by Promega (Madison, Wisconsin) Wizard SV Gel and PCR Cleanup System according to manufacturer’s protocol.

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: .. 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). ..

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Article Title: Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea
    Article Snippet: .. Polymerase chain reactions were performed using Phusion DNA polymerase (Thermo Scientific, Espoo, Finland) on a MyCycler thermocycler for 25 cycles of denaturation at 98 °C for 10 s, annealing at 69 °C for 30 s, and extension at 72 °C for 30 s. The amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Hilden, Germany) and were quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Three independent PCR amplicons obtained from the individual coral metagenomic sample were performed.

    DNA Sequencing:

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). .. The amplimer, flanked by the BamHI and HindIII restriction sites, was cloned into the shuttle vector pMV261 to obtain the pLNA29 plasmid, whose integrity was confirmed by PCR, restriction mapping, and DNA sequencing.

    Sequencing:

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: The groEL gene sequence (GenBank accession number KGP00134.1 ) was obtained from the complete genome sequence of the MOR02 strain ( ) (GenBank accession number JQCV0100000024), and specific primers were used to amplify the complete groEL gene sequence ( ). .. PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture.

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: .. Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: Reporter and viral proteins were transiently expressed in plants from corresponding gene sequences expressed from caulimovirus 35SP‐driven, pROK2‐based binary vectors: vectors expressing the free GFP reporter and CMV (Fny) 2b protein have been described in González et al . ( ); the binary vector expressing PVY P1‐6×‐HCPro (construct P1‐6×‐HCPro) has been described in Tena et al . ( ); the binary vector expressing TBSV P19 suppressor has been described in Canto et al (construct 6×‐2b‐HA), the 2b coding sequence was amplified in sequential polymerase chain reaction (PCR) events with flanking oligos that contained the HA peptide and 6×histidine sequences, resulting in a 2b sequence that had attached, at its 3′ end, an Asc I site plus that of the HA peptide in frame and a Sac I site plus a stop codon at its end, and, at its 5′ end, an Xba I site followed by an ATG, the sequence coding for 6×histidine and a BamH I site in frame with the following sequence coding for 5′ end nucleotides of the 2b sequence. .. All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: Genotyping of the OATP1B1 c. 521 T > C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol
    Article Snippet: In a 20 μl reaction volume containing dNTPs (0.5 mM each), the primer pair (0.5 μM each), 1x Phusion buffer, and 2 units of Phusion DNA polymerase (Thermo Fisher Scientific, Carlsbad, CA), PCR efficiency was compared among reactions containing different amounts of template DNA of 10, 50, 100 or 200 ng. .. It is important to use an optimized amount of template DNA from FFPE tissue to ensure the successfulness of an efficient PCR, which is a prerequisite for sequencing.

    Article Title: Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance
    Article Snippet: Paragraph title: Sequence analysis of target sites in TSWV RNAs ... PCR to amplify TS fragments from segment L or segment M (604 bp or 537 bp, respectively) was performed using oligonucleotide pairs AC‐205/AC‐206 or AC‐209/AC‐210 (Table ), respectively, and Phusion DNA polymerase (ThermoFisher Scientific) with the following program: 30 s at 98°C; 32 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; 10 min at 72°C.

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in . ..

    Article Title: Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins
    Article Snippet: Complementation assays Klebsiella genome regions, containing ExbB , ExbBD , OmpC , FhuA , TonB and FimB gene ORFs along with 5′ non-coding promoter regions, were PCR-amplified from K . quasipneumoniae DSM 28212 genomic DNA with help of Phusion DNA polymerase (Thermofisher Scientific Baltics) and ligated in pJET1.2 (Thermofisher Scientific Baltics). .. After sequencing, cloned fragments were excised with a pair of restriction endonucleases specific for each fragment, ligated in pACYC184 (NEB) and transformed into the respective K . quasipneumoniae mutants.

    Article Title: Role of rhesus macaque IFITM3(2) in simian immunodeficiency virus infection of macaques
    Article Snippet: Paragraph title: DNA sequence analysis of rhIFITM3(2) ... Reaction mixtures contained 100 ng genomic DNA, 50 pmol of each primer, 100 μM dNTP mix, 10% betain, 1x HF synthesis buffer and 1 U Phusion DNA polymerase (ThermoFisher).

    Article Title: Survival of Polyploid hybrid salamander embryos
    Article Snippet: Specific PCR conditions For the lab-reared egg samples, we amplified the D-loop from 1 μl of extracted egg DNA using Phusion DNA polymerase (Thermo Scientific) and 2 μl of mixed 10 μM DL1/007 primer pair [ ] in 25 μl total reaction volumes. .. We submitted samples to the University of Massachusetts Amherst Genomics and Bioinformatics Facility for sequencing from the DL1 primer.

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. The corresponding DNA coding sequences of the NLS from SV40 (MLQPKKKRKV), a nonfunctional mutated nls variant (MLQPNNNNN), an NES from the heat-stable protein kinase inhibitor PKI (NELALKLAGLDINK), a nonfunctional mutated nes variant (NELALKAAGADANK), and a myristoylation motif from CBL1 (MYR) (MGCFHSKAAKEF) were fused to the coding sequence of Rep via PCR amplification.

    Article Title: Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea
    Article Snippet: Paragraph title: 16s rRNA amplification and sequencing ... Polymerase chain reactions were performed using Phusion DNA polymerase (Thermo Scientific, Espoo, Finland) on a MyCycler thermocycler for 25 cycles of denaturation at 98 °C for 10 s, annealing at 69 °C for 30 s, and extension at 72 °C for 30 s. The amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Hilden, Germany) and were quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

    Mutagenesis:

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Subcloning:

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: Escherichia coli strain DH5α was used for subcloning. .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher).

    Purification:

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture. .. The purified 1.65-kb reaction product was digested with the NdeI and EcoRI restriction enzymes and inserted into a pET28 cloning vector previously digested with the same enzymes.

    Article Title: Role of rhesus macaque IFITM3(2) in simian immunodeficiency virus infection of macaques
    Article Snippet: Reaction mixtures contained 100 ng genomic DNA, 50 pmol of each primer, 100 μM dNTP mix, 10% betain, 1x HF synthesis buffer and 1 U Phusion DNA polymerase (ThermoFisher). .. Amplification products were separated on 1% agarose gels, purified and sequenced directly.

    Article Title: Survival of Polyploid hybrid salamander embryos
    Article Snippet: Specific PCR conditions For the lab-reared egg samples, we amplified the D-loop from 1 μl of extracted egg DNA using Phusion DNA polymerase (Thermo Scientific) and 2 μl of mixed 10 μM DL1/007 primer pair [ ] in 25 μl total reaction volumes. .. We cycled each reaction on a Perkin Elmer 9600 thermocycler as follows: initial denaturation at 98 °C for 120 s, followed by 35 cycles of 98 °C:10 s; 55 °C:15 s; and 72 °C:30 s. We confirmed clean amplification by agarose gel electrophoresis and purified the PCR products by Promega (Madison, Wisconsin) Wizard SV Gel and PCR Cleanup System according to manufacturer’s protocol.

    Article Title: Comparative analysis of bacterial communities associated with healthy and diseased corals in the Indonesian sea
    Article Snippet: .. Polymerase chain reactions were performed using Phusion DNA polymerase (Thermo Scientific, Espoo, Finland) on a MyCycler thermocycler for 25 cycles of denaturation at 98 °C for 10 s, annealing at 69 °C for 30 s, and extension at 72 °C for 30 s. The amplicons were purified using QIAGEN PCR purification kit (QIAGEN, Hilden, Germany) and were quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). .. Three independent PCR amplicons obtained from the individual coral metagenomic sample were performed.

    Protein Purification:

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: .. Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). .. The genes for Ml -Fd and sFd-35-ER were cloned into pET-28b to create expression vectors for protein purification.

    Transgenic Assay:

    Article Title: Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance
    Article Snippet: Sequence analysis of target sites in TSWV RNAs The TS sequences from viral progeny of TSWV‐infected transgenic plants were analyzed by RT‐PCR followed by Sanger sequencing. cDNA was obtained from 1–5 μg of total RNA from apical S. lycopersicum leaves using RevertAid reverse transcriptase (RT; ThermoFisher Scientific). .. PCR to amplify TS fragments from segment L or segment M (604 bp or 537 bp, respectively) was performed using oligonucleotide pairs AC‐205/AC‐206 or AC‐209/AC‐210 (Table ), respectively, and Phusion DNA polymerase (ThermoFisher Scientific) with the following program: 30 s at 98°C; 32 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; 10 min at 72°C.

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in . ..

    CRISPR:

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: Paragraph title: Generation and Characterization of CRISPR/Cas9-Mediated Knockout Mutants of S2 -SLF1 ... The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific).

    Polymerase Cycling Assembly:

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: .. These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). ..

    Plasmid Preparation:

    Article Title: Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation
    Article Snippet: PCR was carried out under the following conditions: 30 cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by a final extension step for 10 min at 72°C, amplified with Phusion DNA polymerase (Thermo Fisher Scientific) in a 50-μl reaction mixture. .. The purified 1.65-kb reaction product was digested with the NdeI and EcoRI restriction enzymes and inserted into a pET28 cloning vector previously digested with the same enzymes.

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions
    Article Snippet: Paragraph title: Binary vector constructs ... All PCRs were performed with Phusion™ DNA polymerase (Finnzymes, Keilaranta, Finland).

    Article Title: A flavin-dependent monooxgenase confers resistance to chlorantraniliprole in the diamondback moth, Plutella xylostella
    Article Snippet: Candidate resistance genes were then synthesised (GeneArt, ThermoScientific, USA) and cloned into the pUASTattB plasmid (GenBank: EF362409.1 ). .. The transgenic lines obtained were balanced and the integration of genes confirmed by PCR and sequencing using Phusion DNA polymerase (Thermo, USA) as described previously ( ) with the primers detailed in .

    Article Title: The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells
    Article Snippet: 2.3 Mtb ctpF gene cloning and expression in M. smegmatis The ctpF gene (Rv1997 ) was amplified by PCR from the template genomic DNA of Mtb H37Ra using the primer pair Fcm2Dir/FpmvHis-Rev ( ) and the Phusion DNA Polymerase (Thermo Scientific). .. The amplimer, flanked by the BamHI and HindIII restriction sites, was cloned into the shuttle vector pMV261 to obtain the pLNA29 plasmid, whose integrity was confirmed by PCR, restriction mapping, and DNA sequencing.

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: .. All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. Point mutations were introduced by site-directed mutagenesis using the QuikChange protocol.

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: Paragraph title: Vector design. ... These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher).

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: .. The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific). ..

    Positron Emission Tomography:

    Article Title: A lysin motif effector subverts chitin‐triggered immunity to facilitate arbuscular mycorrhizal symbiosis
    Article Snippet: Protein purification The coding sequence of RiSLM without signal peptide was amplified using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). .. Eco RI and Hin dIII restriction sites were added to the primers (Supporting Information Table ) to facilitate cloning into the pET‐SUMO vector (Champion™ pET SUMO Expression System).

    Article Title: Metalloprotein switches that display chemical-dependent electron transfer in cells
    Article Snippet: These were constructed using Golden Gate DNA assembly of PCR products amplified using Phusion DNA polymerase (Thermo-Fisher). .. The genes for Ml -Fd and sFd-35-ER were cloned into pET-28b to create expression vectors for protein purification.

    Agarose Gel Electrophoresis:

    Article Title: Survival of Polyploid hybrid salamander embryos
    Article Snippet: Specific PCR conditions For the lab-reared egg samples, we amplified the D-loop from 1 μl of extracted egg DNA using Phusion DNA polymerase (Thermo Scientific) and 2 μl of mixed 10 μM DL1/007 primer pair [ ] in 25 μl total reaction volumes. .. We cycled each reaction on a Perkin Elmer 9600 thermocycler as follows: initial denaturation at 98 °C for 120 s, followed by 35 cycles of 98 °C:10 s; 55 °C:15 s; and 72 °C:30 s. We confirmed clean amplification by agarose gel electrophoresis and purified the PCR products by Promega (Madison, Wisconsin) Wizard SV Gel and PCR Cleanup System according to manufacturer’s protocol.

    Knock-Out:

    Article Title: S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen
    Article Snippet: Paragraph title: Generation and Characterization of CRISPR/Cas9-Mediated Knockout Mutants of S2 -SLF1 ... The pGTR plasmid (obtained from Yinong Yang laboratory, Pennsylvania State University) was used as a template to synthesize the tRNA-gRNA fragment (PTG gene) in two separate PCRs using Phusion DNA polymerase (Thermo Scientific).

    Variant Assay:

    Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein
    Article Snippet: All of the constructs were generated by PCR amplification using Phusion DNA polymerase (Thermo Fisher), recombination with the Gateway vector pDONR207 (Thermo Fisher) using the BP Clonase II (Thermo Fisher) reaction, and subsequent transfer to destination vectors using the Gateway LR Clonase II reaction (Thermo Fisher). .. The corresponding DNA coding sequences of the NLS from SV40 (MLQPKKKRKV), a nonfunctional mutated nls variant (MLQPNNNNN), an NES from the heat-stable protein kinase inhibitor PKI (NELALKLAGLDINK), a nonfunctional mutated nes variant (NELALKAAGADANK), and a myristoylation motif from CBL1 (MYR) (MGCFHSKAAKEF) were fused to the coding sequence of Rep via PCR amplification.

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