phusion dna polymerase  (Thermo Fisher)


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    Name:
    Phusion High Fidelity DNA Polymerase 2 U µL
    Description:
    Thermo Scientific Phusion High Fidelity DNA Polymerases set a gold standard for high performance PCR Featuring an error rate 50 fold lower than that of Taq and 6 fold lower than that of Pfu Phusion High Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity Phusion DNA Polymerases offer robust performance with short protocol times even in the presence of PCR inhibitors and generate higher yields with lower enzyme amounts than other DNA polymerase Highlights• High fidelity 52X Taq • Fast PCR due to short extension times 15 30 s kb • Robust performance minimal optimization needed• High yields of PCR products with minimal enzyme amounts• Available as a Green buffer format for direct loading of PCR products on gels F 534S or F 534L Applications• High fidelity PCR• Cloning• Template generation for sequencing• Amplification of difficult GC rich templates• Long range PCR up to 20 kb • Mutagenesis• High throughput PCR• MicroarrayUsing Phusion DNA Polymerases Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases such as Taq DNA polymerases For optimal results use our Tm calculator at www thermofisher com tmcalculator
    Catalog Number:
    f530l
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    Applications:
    High Fidelity PCR|PCR|PCR & Real-Time PCR
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    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher phusion dna polymerase
    ( A ) Template hybridization, extension, and denaturation on the flowcell surface. Templates are prepared so as to possess tails that are complementary to primers on the flowcell surface. This allows one end of a template strand to hybridize to a flowcell primer. Flowcell primers are extended by <t>Phusion</t> <t>DNA</t> polymerase (Thermo Scientific), resulting in a reverse complementary copy of the original template strand, which is covalently attached to the flowcell surface. The original template strand is then removed by flushing 0.1 M NaOH though the flowcell. ( B ) Cluster amplification. The free end of the tethered reverse complementary copy of the original template strand can anneal to the other type of flowcell primer, forming a bridge. The flowcell primer is extended by Bst polymerase, in an isothermal reaction, which generates a double-stranded product. Formamide is used to denature these strands, which can then anneal to other primers on the flowcell surface, which extend in the next cycle. In this way, repeated cycles of extension and denaturation result in a cluster of strands, all of which are derived from a single template strand.
    Thermo Scientific Phusion High Fidelity DNA Polymerases set a gold standard for high performance PCR Featuring an error rate 50 fold lower than that of Taq and 6 fold lower than that of Pfu Phusion High Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity Phusion DNA Polymerases offer robust performance with short protocol times even in the presence of PCR inhibitors and generate higher yields with lower enzyme amounts than other DNA polymerase Highlights• High fidelity 52X Taq • Fast PCR due to short extension times 15 30 s kb • Robust performance minimal optimization needed• High yields of PCR products with minimal enzyme amounts• Available as a Green buffer format for direct loading of PCR products on gels F 534S or F 534L Applications• High fidelity PCR• Cloning• Template generation for sequencing• Amplification of difficult GC rich templates• Long range PCR up to 20 kb • Mutagenesis• High throughput PCR• MicroarrayUsing Phusion DNA Polymerases Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases such as Taq DNA polymerases For optimal results use our Tm calculator at www thermofisher com tmcalculator
    https://www.bioz.com/result/phusion dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 134 article reviews
    Price from $9.99 to $1999.99
    phusion dna polymerase - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Improved Protocols for Illumina Sequencing"

    Article Title: Improved Protocols for Illumina Sequencing

    Journal: Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]

    doi: 10.1002/0471142905.hg1802s62

    ( A ) Template hybridization, extension, and denaturation on the flowcell surface. Templates are prepared so as to possess tails that are complementary to primers on the flowcell surface. This allows one end of a template strand to hybridize to a flowcell primer. Flowcell primers are extended by Phusion DNA polymerase (Thermo Scientific), resulting in a reverse complementary copy of the original template strand, which is covalently attached to the flowcell surface. The original template strand is then removed by flushing 0.1 M NaOH though the flowcell. ( B ) Cluster amplification. The free end of the tethered reverse complementary copy of the original template strand can anneal to the other type of flowcell primer, forming a bridge. The flowcell primer is extended by Bst polymerase, in an isothermal reaction, which generates a double-stranded product. Formamide is used to denature these strands, which can then anneal to other primers on the flowcell surface, which extend in the next cycle. In this way, repeated cycles of extension and denaturation result in a cluster of strands, all of which are derived from a single template strand.
    Figure Legend Snippet: ( A ) Template hybridization, extension, and denaturation on the flowcell surface. Templates are prepared so as to possess tails that are complementary to primers on the flowcell surface. This allows one end of a template strand to hybridize to a flowcell primer. Flowcell primers are extended by Phusion DNA polymerase (Thermo Scientific), resulting in a reverse complementary copy of the original template strand, which is covalently attached to the flowcell surface. The original template strand is then removed by flushing 0.1 M NaOH though the flowcell. ( B ) Cluster amplification. The free end of the tethered reverse complementary copy of the original template strand can anneal to the other type of flowcell primer, forming a bridge. The flowcell primer is extended by Bst polymerase, in an isothermal reaction, which generates a double-stranded product. Formamide is used to denature these strands, which can then anneal to other primers on the flowcell surface, which extend in the next cycle. In this way, repeated cycles of extension and denaturation result in a cluster of strands, all of which are derived from a single template strand.

    Techniques Used: Hybridization, Amplification, Derivative Assay

    2) Product Images from "Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments"

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl635

    Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.
    Figure Legend Snippet: Characteristics and applications of the USER technique. ( a ) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum ® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo ® C x Hotstart DNA polymerase. ( b ) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus -specific USER compatible vector, pNB1u. H 2 O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. ( c ) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

    Techniques Used: Functional Assay, Expressing, Plasmid Preparation, Injection, Fluorescence

    3) Product Images from "Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete"

    Article Title: Suggested Role for G4 DNA in Recombinational Switching at the Antigenic Variation Locus of the Lyme Disease Spirochete

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057792

    Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.
    Figure Legend Snippet: Precise excision of the vlsE variable region during PCR amplification. A) Schematic showing the variable, constant and 17 bp direct repeats of the vlsE gene. The location of PCR primers used for amplification are shown by arrows below the constant regions. B) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using three different templates and two different primer sets. The templates used were B. burgdorferi B31 5A4 genomic DNA [29] pMBL20, a plasmid carrying the vlsE gene [51] ; the 776 bp or 935 bp PCR products resulting from PCR amplification. The asterisks indicate smaller discrete bands observed in lanes 1–3 and 4–6. M denotes a 100 bp molecular weight ladder marker. PCR samples were run on a 1.2% agarose gel in 1X TAE buffer at 80 V for 1.2 hours. C) Characterization of precise deletions in vlsE . The first entry on the left side of the panel shows the sequence obtained from direct sequencing of the PCR product obtained with either B248 and B249 or B1701 and 1702. The remainder of the lineup shows sequence generated with 10 randomly selected E. coli transformants. The transformants were generated by cloning the 223 bp truncated PCR product generated in vitro with primers B248 and B249. The fragment was gel-excised and cloned into pJET1.2/blunt vector (Fermentas). The alignment shows that all the sequenced vlsE inserts had a precise excision of the 570 bp variable region. D) Schematic showing the precise excision of the vlsE variable region that occurred during PCR amplification.

    Techniques Used: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis, Plasmid Preparation, Molecular Weight, Marker, Sequencing, Generated, Clone Assay, In Vitro

    Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region. A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C ) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2 .
    Figure Legend Snippet: Affect of mutations in the 17 bp direct repeats on precise excision of the vlsE variable region. A) DNA sequences of the wild-type 17 bp direct repeat (DR) and a mutant 17 bp direct repeat (DR*) used in this study. Mutated bases are highlighted in red. B) Schematic showing the plasmid templates carrying wild-type DRs and a mutant DR at the left, right or both sides of the variable region. C ) An ethidium bromide-stained agarose gel showing amplification of a portion of vlsE with Phusion DNA polymerase using the templates shown in Panel B with the indicated primers. Gel electrophoresis conditions were as noted in Fig. 2 .

    Techniques Used: Mutagenesis, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Amplification, Nucleic Acid Electrophoresis

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments
    Article Snippet: .. PCR conditions PCR with the following DNA polymerases: HotMaster™ Taq DNA Polymerase (Eppendorf), Platinum® Taq DNA Polymerase (Invitrogen), Pwo DNA Polymerase (Roche), Phusion™ DNA Polymerase (Finnzymes), and PfuTurbo® Cx Hotstart DNA polymerase (PfuCx) (Stratagene) was performed according to manufacturers' instructions on pBAD-TOPO® (Invitrogen) containing the gene, At5g43440 (Accession no. AY143873). ..

    Article Title: Production of in vitro amplified DNA pseudolibraries and high-throughput cDNA target amplification
    Article Snippet: .. Target amplifications When the novel Phusion DNA polymerase PCR mixture (Finnzymes) became available, we decided to compare its performance with that of the most successful PCR set up known to us. ..

    Article Title: Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results
    Article Snippet: .. Taq polymerase A range of 13 high fidelity, regular, economy and premium Taq polymerase enzymes were selected: Biotaq® (Bioline, London, UK), FastStart® High Fidelity PCR System (Roche, Mannheim, Germany), AmpliTaq Gold® (Applied Biosystems, Warrington, UK), HotStarTaq® DNA Polymerase (Qiagen, Hilden, Gernamy), Phusion® High Fidelity DNA Polymerase (Finnzymes, Espoo, Finland), Taq DNA Polymerase (Roche, Maylan, France), i-MaxTM II DNA Polymerase (iNtRON Biotechnology, Seongnam, Korea), KAPA HiFi™ (Kapa Biosystems, Boston, USA), OneTaq ™ DNA Polymerase (New England Biolabs, Hitchin, UK), Vent® DNA Polymerase (New England Biolabs, Hitchin, UK), Deep Vent® DNA Polymerase (New England Biolabs, Hitchin, UK), Pwo® DNA Polymerase (Roche, Maylan, France) and Velocity DNA Polymerase (Bioline, London, UK) (abbreviated names in ). ..

    Article Title: Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants
    Article Snippet: .. The following DNA polymerases were tested: Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, USA, cat #F530S), Ex-Taq DNA polymerase (Clontech, USA, cat #RR001A), Taq DNA polymerase (SibEnzyme, Russia, cat #E331), Emerald Amp GT PCR Master Mix (Clontech, USA, cat #RR310A). .. 10 ng of genomic DNA and 10 μM of each primer were used in all PCR reactions; all other components of PCR reactions were added according to the manufacturer’s suggested protocol for each individual polymerase.

    Isolation:

    Article Title: Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes
    Article Snippet: .. Determination of the relative amount of circular plasmid DNA in transformed pneumococcal cells by iPCR The plasmidic DNA (pDNA) present in the gDNA isolated from the transformed pneumococcal cells was used as template to perform an inverse PCR protocol with a primer set of divergent oligonucleotides. iPCR was performed using the Phusion High Fidelity (HF) (Thermo Scientific) DNA polymerase. .. Amplification reactions (20 μl) contained 0.7 ng of gDNA and 0.5 μM of the specific forward and reverse primers (Table ).

    Transformation Assay:

    Article Title: Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes
    Article Snippet: .. Determination of the relative amount of circular plasmid DNA in transformed pneumococcal cells by iPCR The plasmidic DNA (pDNA) present in the gDNA isolated from the transformed pneumococcal cells was used as template to perform an inverse PCR protocol with a primer set of divergent oligonucleotides. iPCR was performed using the Phusion High Fidelity (HF) (Thermo Scientific) DNA polymerase. .. Amplification reactions (20 μl) contained 0.7 ng of gDNA and 0.5 μM of the specific forward and reverse primers (Table ).

    Inverse PCR:

    Article Title: Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes
    Article Snippet: .. Determination of the relative amount of circular plasmid DNA in transformed pneumococcal cells by iPCR The plasmidic DNA (pDNA) present in the gDNA isolated from the transformed pneumococcal cells was used as template to perform an inverse PCR protocol with a primer set of divergent oligonucleotides. iPCR was performed using the Phusion High Fidelity (HF) (Thermo Scientific) DNA polymerase. .. Amplification reactions (20 μl) contained 0.7 ng of gDNA and 0.5 μM of the specific forward and reverse primers (Table ).

    Plasmid Preparation:

    Article Title: Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes
    Article Snippet: .. Determination of the relative amount of circular plasmid DNA in transformed pneumococcal cells by iPCR The plasmidic DNA (pDNA) present in the gDNA isolated from the transformed pneumococcal cells was used as template to perform an inverse PCR protocol with a primer set of divergent oligonucleotides. iPCR was performed using the Phusion High Fidelity (HF) (Thermo Scientific) DNA polymerase. .. Amplification reactions (20 μl) contained 0.7 ng of gDNA and 0.5 μM of the specific forward and reverse primers (Table ).

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    Thermo Fisher dna polymerases
    Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) <t>Phusion</t> High-Fidelity <t>DNA</t> polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.
    Dna Polymerases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerases/product/Thermo Fisher
    Average 99 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    dna polymerases - by Bioz Stars, 2020-08
    99/100 stars
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    84
    Thermo Fisher phusion high fidelity dna polymeraseeach pcr amplification
    <t>PCR</t> performance of Herculase® II Fusion <t>DNA</t> polymerase and <t>Phusion™</t> High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.
    Phusion High Fidelity Dna Polymeraseeach Pcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymeraseeach pcr amplification/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity dna polymeraseeach pcr amplification - by Bioz Stars, 2020-08
    84/100 stars
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    99
    Thermo Fisher mutagenesis pcr
    Expression of poplar root TPS genes and emission of major TPS products. Expression of TPS genes was analyzed using <t>qRT-PCR.</t> TPS expression and emission of monoterpenes are displayed for M. melolontha -damaged (herb) and undamaged (ctr) roots from P. trichocarpa and P. nigra . Means ± SE are shown (n = 8). Asterisks indicate statistical significance in Student’s t-tests or from Mann-Whitney Rank Sum Tests. PtTPS13 ( P ≤ 0.001, T = 36.00); PtTPS21 ( P = 0.209, T = 42.00); <t>PtTPS16</t> ( P = 0.195, T = 55.00); PnTPS4 ( P ≤ 0.001, T = 36.00); PnTPS1 ( P ≤ 0.001, T = 36.00); P. trichocarpa : 1.8-cineole ( P = 0.048, t = 2.168); camphene ( P = 0.061, t = −2.042); p -cymene ( P = 0.458, t = −0.764); P. nigra : 1.8-cineole ( P = 0.578, t = −0.570); camphene ( P ≤ 0.001, T = 37.00).
    Mutagenesis Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mutagenesis pcr/product/Thermo Fisher
    Average 99 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    mutagenesis pcr - by Bioz Stars, 2020-08
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    Image Search Results


    Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Journal: BioNanoScience

    Article Title: Selection of efficient Taq DNA polymerase to optimize T-DNA genotyping method for rapid detection of mutant Arabidopsis thaliana plants

    doi: 10.1007/s12668-016-0253-6

    Figure Lengend Snippet: Genotyping of Arabidopsis thaliana plants from SALK_100012 line with (a) Phusion High-Fidelity DNA polymerase; (b) Ex-Taq DNA polymerase; (c) Taq DNA polymerase; (d) Emerald Amp GT PCR Master Mix. WT - DNA from wild-type Arabidopsis plant (positive control for wild-type PCR band); 1–8 - individual plants from SALK_100012 line. Molecular weight DNA standards are shown on each side. Loading order for each DNA sample: PCR reaction for wild-type allele, PCR reaction for T-DNA mutant allele.

    Article Snippet: The following DNA polymerases were tested: Phusion High-Fidelity DNA polymerase (ThermoFisher Scientific, USA, cat #F530S), Ex-Taq DNA polymerase (Clontech, USA, cat #RR001A), Taq DNA polymerase (SibEnzyme, Russia, cat #E331), Emerald Amp GT PCR Master Mix (Clontech, USA, cat #RR310A).

    Techniques: Polymerase Chain Reaction, Positive Control, Molecular Weight, Mutagenesis

    The presence of CopG in the recipient cell impairs repopulation of the pMV158 replicon by decreasing the plasmid replication rate. Changes in the fraction of transformants retaining newly-acquired pLS1 cop7 (a copy-up pMV158 derivative) were analyzed in pneumococcal strains that either harbor a high dosage of active copG (A) or a truncated version of copG (B) . The experimental loss rate ( L ex ) of pLS1 cop7 was calculated from the slope of the linear regression model of the plot of the experimental values according to Equation (2) (red circles and lines). T 0 and T are, respectively, the fractions of transformants ab initio and after n generations. The graphs in (C,D) show the impact of CopG on the kinetics of pLS1 cop7 repopulation. A qPCR approach was used to calculate the variation in the copy number of a specific amplicon of the incoming pLS1 cop7 plasmid relative to the chromosome during the growth of the total bacterial population containing pCGA3 (C) or pCGA30 (D) as resident plasmid for 150 min after transformation (left y-axis; blue circle and lines). The pLS1 cop7 replication rate ( R value), defined as the ratio of plasmid to gDNA duplications, was calculated at different time intervals following transformation of pneumococcal cells harboring pCGA3 (C) or pCGA30 (D) . Determination of R was based on the iPCR data of the in vivo plasmid amplification (black circles and lines) and its value (right y-axis) was calculated according to Equation (10). Discontinuous horizontal line in graphs of (C,D) denotes an R value of 1, which characterizes the steady-state plasmid replication. The mean (symbols) and standard deviation (error bars) of all the experimental points in the graphs of (C,D) are displayed. Panels (E,F) show the iPCR analysis of the gDNA samples obtained at the indicated times after transformation of pneumococcal cells carrying pCGA3 and pCGA30, respectively, with pLS1 cop7 . iPCR assays were carried out by using a pair of divergent primers specific for the pMV158 replicon (Table 3 and G ) and the Phusion DNA polymerase. Lane M, DNA molecular weight standard (NZYDNA ladder III; NZYTECH). Note that lanes M are the same in (E,F) because, in fact, both images of these panels arise from the same gel. Dividing lines in (E) indicate grouping of different parts of the same gel. The original image of the gel used for (E,F) composition is shown in Figure S2 . A schematic representation of pLS1 cop7 displaying the plasmid regions complementary to the divergent primers is shown in (G) . Genes copG, repB , and tetL , as well as the dso region, are indicated.

    Journal: Frontiers in Microbiology

    Article Title: Successful Establishment of Plasmids R1 and pMV158 in a New Host Requires the Relief of the Transcriptional Repression of Their Essential rep Genes

    doi: 10.3389/fmicb.2017.02367

    Figure Lengend Snippet: The presence of CopG in the recipient cell impairs repopulation of the pMV158 replicon by decreasing the plasmid replication rate. Changes in the fraction of transformants retaining newly-acquired pLS1 cop7 (a copy-up pMV158 derivative) were analyzed in pneumococcal strains that either harbor a high dosage of active copG (A) or a truncated version of copG (B) . The experimental loss rate ( L ex ) of pLS1 cop7 was calculated from the slope of the linear regression model of the plot of the experimental values according to Equation (2) (red circles and lines). T 0 and T are, respectively, the fractions of transformants ab initio and after n generations. The graphs in (C,D) show the impact of CopG on the kinetics of pLS1 cop7 repopulation. A qPCR approach was used to calculate the variation in the copy number of a specific amplicon of the incoming pLS1 cop7 plasmid relative to the chromosome during the growth of the total bacterial population containing pCGA3 (C) or pCGA30 (D) as resident plasmid for 150 min after transformation (left y-axis; blue circle and lines). The pLS1 cop7 replication rate ( R value), defined as the ratio of plasmid to gDNA duplications, was calculated at different time intervals following transformation of pneumococcal cells harboring pCGA3 (C) or pCGA30 (D) . Determination of R was based on the iPCR data of the in vivo plasmid amplification (black circles and lines) and its value (right y-axis) was calculated according to Equation (10). Discontinuous horizontal line in graphs of (C,D) denotes an R value of 1, which characterizes the steady-state plasmid replication. The mean (symbols) and standard deviation (error bars) of all the experimental points in the graphs of (C,D) are displayed. Panels (E,F) show the iPCR analysis of the gDNA samples obtained at the indicated times after transformation of pneumococcal cells carrying pCGA3 and pCGA30, respectively, with pLS1 cop7 . iPCR assays were carried out by using a pair of divergent primers specific for the pMV158 replicon (Table 3 and G ) and the Phusion DNA polymerase. Lane M, DNA molecular weight standard (NZYDNA ladder III; NZYTECH). Note that lanes M are the same in (E,F) because, in fact, both images of these panels arise from the same gel. Dividing lines in (E) indicate grouping of different parts of the same gel. The original image of the gel used for (E,F) composition is shown in Figure S2 . A schematic representation of pLS1 cop7 displaying the plasmid regions complementary to the divergent primers is shown in (G) . Genes copG, repB , and tetL , as well as the dso region, are indicated.

    Article Snippet: Determination of the relative amount of circular plasmid DNA in transformed pneumococcal cells by iPCR The plasmidic DNA (pDNA) present in the gDNA isolated from the transformed pneumococcal cells was used as template to perform an inverse PCR protocol with a primer set of divergent oligonucleotides. iPCR was performed using the Phusion High Fidelity (HF) (Thermo Scientific) DNA polymerase.

    Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction, Amplification, Transformation Assay, In Vivo, Standard Deviation, Molecular Weight

    PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.

    Journal: Journal of Nematology

    Article Title: Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease

    doi: 10.21307/jofnem-2020-016

    Figure Lengend Snippet: PCR performance of Herculase® II Fusion DNA polymerase and Phusion™ High-Fidelity DNA Polymerase. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. 1, 2, 3 and 4: Herculase® II Fusion DNA polymerase; 5, 6, 7 and 8: Phusion™ High Fidelity PCR System; NC: negative control, respectively.

    Article Snippet: Phusion™ High-Fidelity DNA polymeraseEach PCR amplification with Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific, Waltham, MA) was carried out in a 25 μl of mixture containing Phusion™ High-Fidelity DNA Polymerase (2 units/μl) 0.25 μl, 5× reaction buffer 5 μl, dNTP (2.5 mM each) 0.5 μl, Template DNA 2 μl, forward primer 18S-CL-F3 (10 μm) 1.25 μl, reverse primer 28S-CL-R (10 μm) 1.25 μl, DMSO 0.25 μl, and molecular biology grade water (Sigma-Aldrich, St Louis, MO) 14.5 μl.

    Techniques: Polymerase Chain Reaction, Negative Control

    Expression of poplar root TPS genes and emission of major TPS products. Expression of TPS genes was analyzed using qRT-PCR. TPS expression and emission of monoterpenes are displayed for M. melolontha -damaged (herb) and undamaged (ctr) roots from P. trichocarpa and P. nigra . Means ± SE are shown (n = 8). Asterisks indicate statistical significance in Student’s t-tests or from Mann-Whitney Rank Sum Tests. PtTPS13 ( P ≤ 0.001, T = 36.00); PtTPS21 ( P = 0.209, T = 42.00); PtTPS16 ( P = 0.195, T = 55.00); PnTPS4 ( P ≤ 0.001, T = 36.00); PnTPS1 ( P ≤ 0.001, T = 36.00); P. trichocarpa : 1.8-cineole ( P = 0.048, t = 2.168); camphene ( P = 0.061, t = −2.042); p -cymene ( P = 0.458, t = −0.764); P. nigra : 1.8-cineole ( P = 0.578, t = −0.570); camphene ( P ≤ 0.001, T = 37.00).

    Journal: Scientific Reports

    Article Title: The occurrence and formation of monoterpenes in herbivore-damaged poplar roots

    doi: 10.1038/s41598-018-36302-6

    Figure Lengend Snippet: Expression of poplar root TPS genes and emission of major TPS products. Expression of TPS genes was analyzed using qRT-PCR. TPS expression and emission of monoterpenes are displayed for M. melolontha -damaged (herb) and undamaged (ctr) roots from P. trichocarpa and P. nigra . Means ± SE are shown (n = 8). Asterisks indicate statistical significance in Student’s t-tests or from Mann-Whitney Rank Sum Tests. PtTPS13 ( P ≤ 0.001, T = 36.00); PtTPS21 ( P = 0.209, T = 42.00); PtTPS16 ( P = 0.195, T = 55.00); PnTPS4 ( P ≤ 0.001, T = 36.00); PnTPS1 ( P ≤ 0.001, T = 36.00); P. trichocarpa : 1.8-cineole ( P = 0.048, t = 2.168); camphene ( P = 0.061, t = −2.042); p -cymene ( P = 0.458, t = −0.764); P. nigra : 1.8-cineole ( P = 0.578, t = −0.570); camphene ( P ≤ 0.001, T = 37.00).

    Article Snippet: In vitro mutagenesis For site-directed mutagenesis, 100 ng pET100/D-TOPO® vector containing the N-terminal truncated ORF of PtTPS16 was used as template in a mutagenesis PCR (18 cycles, Phusion® High-Fidelity DNA Polymerase (ThermoFisher Scientific), according to manufacturer’s instructions.

    Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY