phusion dna polymerase Thermo Fisher Search Results


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  • 95
    New England Biolabs phusion high fidelity pcr master mix
    Phusion High Fidelity Pcr Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion high fidelity dna polymerase
    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial <t>DNA</t> (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of <t>Phusion,</t> Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .
    Phusion High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion u hot start dna polymerase
    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial <t>DNA</t> (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of <t>Phusion,</t> Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .
    Phusion U Hot Start Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher high fidelity phushion dna polymerase
    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial <t>DNA</t> (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of <t>Phusion,</t> Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .
    High Fidelity Phushion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion dna polymerase reaction mix
    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial <t>DNA</t> (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of <t>Phusion,</t> Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .
    Phusion Dna Polymerase Reaction Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher heat stable phusion dna polymerase
    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial <t>DNA</t> (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of <t>Phusion,</t> Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .
    Heat Stable Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher pcr amplification
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    Pcr Amplification, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 52385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher high fidelity f 530 phusion dna polymerase
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    High Fidelity F 530 Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher high fidelity phusion flash dna polymerase
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    High Fidelity Phusion Flash Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mutant strains phusion dna polymerase
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    Mutant Strains Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher plasmid constructs phusion dna polymerase
    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the <t>3C</t> assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C <t>PCR</t> amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).
    Plasmid Constructs Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion hot start dna polymerases
    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand <t>DNA</t> molecules. <t>Phusion</t> DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Phusion Hot Start Dna Polymerases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific phusion high fidelity dna polymerase
    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand <t>DNA</t> molecules. <t>Phusion</t> DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Phusion High Fidelity Dna Polymerase, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 96/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion hot start ii high fidelity dna polymerase
    Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the <t>Phusion</t> HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of <t>DNA</t> bands in DNA marker (lane M) are indicated on the left.
    Phusion Hot Start Ii High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1503 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher two step amplification phusion flash dna polymerase
    Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the <t>Phusion</t> HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of <t>DNA</t> bands in DNA marker (lane M) are indicated on the left.
    Two Step Amplification Phusion Flash Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion dna polymerase master mix
    Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the <t>Phusion</t> HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of <t>DNA</t> bands in DNA marker (lane M) are indicated on the left.
    Phusion Dna Polymerase Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3818 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion dna polymerase i
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Phusion Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher taq dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 36191 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion green hot start ii high fidelity dna polymerase kit
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    Phusion Green Hot Start Ii High Fidelity Dna Polymerase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher phusion taq dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher pfu polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher phusion green hot start ii high fidelity dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher phusion pfu dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher phusion dna polymerse
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher tgcagcctaatcaccgc oligo glsrna 17 rv gtgcagggtccgaggt phusion highfidelity dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher 5x phusion dna polymerase buffer
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher phusion high fidelity pcr kit
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Thermo Fisher full length env genes phusion hotstart ii hi fidelity dna polymerase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
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    Image Search Results


    Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .

    Journal: Scientific Reports

    Article Title: Effect of the enzyme and PCR conditions on the quality of high-throughput DNA sequencing results

    doi: 10.1038/srep08056

    Figure Lengend Snippet: Proportion of correct reads for the three genetic systems (simple: a single allele per individual, squares ; medium: two alleles, circles ; and complex: multiple alleles, triangles ) using standard PCR conditions ( open ) and modified PCR conditions to reduce chimera formation ( gray ). The size of the shape is indicative of the number of reads (see legend). All enzymes yielded at least 50% correct reads in the simplest system, mitochondrial DNA (Test 1; open squares). Some enzymes only worked for a given set of conditions (cycling conditions/genetic system). A group of enzymes consisting of Phusion, Gold and FastStart yielded a high proportion of correct reads cosistently accross all conditions. Others, such as Roche Taq, HotStar and Biotaq, yielded a low percent of correct reads for the more complex systems (MHC class I and MHC class II). Abbreviations as defined in Table 1 .

    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 ).

    Techniques: Polymerase Chain Reaction, Modification

    LINE-1 insertion in intron 2 of AHR in Irish Wolfhounds. Southern blot analysis (A) with HindIII (lanes 1–4) and PstI (lanes 5–8) digested genomic DNA. Digestion with HindIII resulted in an increase in size of about 2000 bp in Irish wolfhounds (lanes 1 and 2) compared to healthy beagles (lanes 3 and 4). Using PstI as restriction enzyme results in a small increase in size of approximately 400 bp in Irish wolfhounds (lanes 5 and 6) compared to healthy beagles (lanes 7 and 8). The observed size differences are smaller than the actual rearrangement due to the presence of restriction enzyme sites in the rearranged DNA fragment. Gel electrophoresis of PCR products (B) with forward primer in exon 2 and reverse primer downstream intron 2. The use of Phusion ™ Hot Start High-Fidelity DNA Polymerase in combination with GC-buffer was required to amplify this fragment. An increase in intron size was detected in all used gDNA samples of Irish wolfhounds. Lane 1∶1 kb DNA ladder (Promega, Leiden, the Netherlands); 2: normal control; 3–5: genomic DNA of Irish wolfhounds; 6: negative control; 7∶100 bp DNA ladder (Promega). A 6298 bp long LINE-1 insertion named L1-Y_CF (C) is located 63 bp downstream of exon2. It contains a 13 bp long duplication site specific for LINE-1 insertions. In both open reading frames of this retrotransposon deletions were detected causing frame shifts and premature stop codons. Restriction sites were found for HindIII at 1908 bp and for PstI at 447 bp.

    Journal: PLoS ONE

    Article Title: Altered Subcellular Localization of Heat Shock Protein 90 Is Associated with Impaired Expression of the Aryl Hydrocarbon Receptor Pathway in Dogs

    doi: 10.1371/journal.pone.0057973

    Figure Lengend Snippet: LINE-1 insertion in intron 2 of AHR in Irish Wolfhounds. Southern blot analysis (A) with HindIII (lanes 1–4) and PstI (lanes 5–8) digested genomic DNA. Digestion with HindIII resulted in an increase in size of about 2000 bp in Irish wolfhounds (lanes 1 and 2) compared to healthy beagles (lanes 3 and 4). Using PstI as restriction enzyme results in a small increase in size of approximately 400 bp in Irish wolfhounds (lanes 5 and 6) compared to healthy beagles (lanes 7 and 8). The observed size differences are smaller than the actual rearrangement due to the presence of restriction enzyme sites in the rearranged DNA fragment. Gel electrophoresis of PCR products (B) with forward primer in exon 2 and reverse primer downstream intron 2. The use of Phusion ™ Hot Start High-Fidelity DNA Polymerase in combination with GC-buffer was required to amplify this fragment. An increase in intron size was detected in all used gDNA samples of Irish wolfhounds. Lane 1∶1 kb DNA ladder (Promega, Leiden, the Netherlands); 2: normal control; 3–5: genomic DNA of Irish wolfhounds; 6: negative control; 7∶100 bp DNA ladder (Promega). A 6298 bp long LINE-1 insertion named L1-Y_CF (C) is located 63 bp downstream of exon2. It contains a 13 bp long duplication site specific for LINE-1 insertions. In both open reading frames of this retrotransposon deletions were detected causing frame shifts and premature stop codons. Restriction sites were found for HindIII at 1908 bp and for PstI at 447 bp.

    Article Snippet: The thermal cycling protocol consisted of a 5-min denaturation step at 95°C, 35 cycles of 30 s at 95°C, 30 s at annealing temperature, 30 s at 72°C, and a final elongation step at 72°C for 10 min. For amplification of fragments exceeding 2000 bp, Phusion ™ Hot Start High-Fidelity DNA Polymerase (Thermo Scientific, Lafayette, US) was used.

    Techniques: Southern Blot, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Negative Control

    The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the 3C assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C PCR amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).

    Journal: The Journal of Biological Chemistry

    Article Title: A Novel Intronic Peroxisome Proliferator-activated Receptor ? Enhancer in the Uncoupling Protein (UCP) 3 Gene as a Regulator of Both UCP2 and -3 Expression in Adipocytes *

    doi: 10.1074/jbc.M110.120584

    Figure Lengend Snippet: The PPARγ binding site in Ucp3 intron 1 loops out to specifically interact with the Ucp2 promoter and 5′-region in adipocytes. A , schematic representation of the mouse Ucp3 and -2 loci showing the position of the +1950 and +400 PPARγ/RXR bindings sites in the Ucp3 and Ucp2 intron 1, respectively ( solid vertical lines ). Sites recognized by the restriction enzyme Csp6I used in the 3C assay are indicated by punctuated lines , and arrows mark the positions of the primers used in the 3C analyses. B , agarose gel picture showing the 3C PCR amplicons generated with the BF-IF primer combination that detects looping between the bait (restriction fragment covering the +1950 PPARγ/RXR binding site in intron 1 of the Ucp3 gene) and the interactor (the restriction fragment covering the Ucp2 promoter and 5′-region, including the +400 PPARγ/RXR bindings site). C , agarose gel picture showing the 3C PCR amplicons generated with the indicated primer combinations (see A ) designed to detect looping between the bait and the genomic regions immediately up- or downstream of the interactor fragment. Lanes are as follows: marker ( M ), no template control ( NTC ), Csp6I-digested 3T3-L1 genomic DNA ( GD ), standard curve of control template ( 2–16 fg ), 3T3-L1 adipocytes day 6 ( d6 ), and non-cross-linked ligated control ( NCLC ).

    Article Snippet: Control fragments for the evaluation of 3C primers were generated by PCR amplification (Phusion hot start high-fidelity DNA polymerase, Finnzymes) of the regions flanking the Csp6I restriction sites at Ucp3 intron 1, the 5′-end of Ucp2 , and the regions upstream and downstream of the latter.

    Techniques: Binding Assay, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Generated, Marker

    ( A ) Schematic of the gene targeting strategy to insert a GFP-Luc reporter gene under control of the hsp70 promoter in the pfs47 locus, showing the genomic locus of the wild-type (WT) and gene deletion mutants ∆pf47 containing the GFP-luciferase ORF before and after removal of the hdhfr resistance marker. P1, P2 : primers MWV300 and MWV301, respectively, used for PCR analysis; X ( XcmI ), N ( NcoI ): restriction sites used for conformation of LR-PCR analysis; pf47 : pf47 locus; hrp: histidine rich protein; cam: calmodulin; hsp: heat shock protein; hdhfr : human dihydrodrofolate reductase coding region; GFP-Luc : GFP-luciferase fusion protein; fcu: cytosine deaminase/uracil phosphoribosyl transferase; pbdt: P. berghei dhfr transcription termination region; kb: kilo basepairs. ( B ) Verification of the integration site by PCR and restriction digestion. Genomic DNA fragments from the parental NF54 strain (WT), intermediate transgene NF54-HGL-hDHFR and final transgene NF54-HGL were amplified by PCR using primers MWV300 and MWV301 (P1 and P2) and analysed by DNA gel electrophoresis. Where indicated, the PCR fragment was subjected to restriction digestion with XcmI/NcoI (X/N) ( C ) Luminescence intensity (relative light units, RLU) as a function of cell number. The figure shows a comparison between uninfected red blood cells (RBC), immature gametocytes (Day 8 GCT), mature gametocytes (Day 14 GCT) and asexual blood stage parasites. ( D–G ) FLP-FRT mediated excision of the selection marker restores sensitivity to antifolates. Drug sensitivity analysis of WT, ∆Pf47GFP-luc-hdhfr and ∆Pf47GFP-luc to DHA ( D ), Chloroquine ( E ), Pyrimethamine ( F ), and WR99210 ( G ) in a dose dependent asexual growth inhibition assay using pLDH enzyme activity as a readout. Depicted data represents a triplicate measurement and was normalized to the MIN (10 μM DHA) and MAX (0.1% DMSO) controls. Error bars indicate standard deviations.

    Journal: Scientific Reports

    Article Title: A semi-automated luminescence based standard membrane feeding assay identifies novel small molecules that inhibit transmission of malaria parasites by mosquitoes

    doi: 10.1038/srep18704

    Figure Lengend Snippet: ( A ) Schematic of the gene targeting strategy to insert a GFP-Luc reporter gene under control of the hsp70 promoter in the pfs47 locus, showing the genomic locus of the wild-type (WT) and gene deletion mutants ∆pf47 containing the GFP-luciferase ORF before and after removal of the hdhfr resistance marker. P1, P2 : primers MWV300 and MWV301, respectively, used for PCR analysis; X ( XcmI ), N ( NcoI ): restriction sites used for conformation of LR-PCR analysis; pf47 : pf47 locus; hrp: histidine rich protein; cam: calmodulin; hsp: heat shock protein; hdhfr : human dihydrodrofolate reductase coding region; GFP-Luc : GFP-luciferase fusion protein; fcu: cytosine deaminase/uracil phosphoribosyl transferase; pbdt: P. berghei dhfr transcription termination region; kb: kilo basepairs. ( B ) Verification of the integration site by PCR and restriction digestion. Genomic DNA fragments from the parental NF54 strain (WT), intermediate transgene NF54-HGL-hDHFR and final transgene NF54-HGL were amplified by PCR using primers MWV300 and MWV301 (P1 and P2) and analysed by DNA gel electrophoresis. Where indicated, the PCR fragment was subjected to restriction digestion with XcmI/NcoI (X/N) ( C ) Luminescence intensity (relative light units, RLU) as a function of cell number. The figure shows a comparison between uninfected red blood cells (RBC), immature gametocytes (Day 8 GCT), mature gametocytes (Day 14 GCT) and asexual blood stage parasites. ( D–G ) FLP-FRT mediated excision of the selection marker restores sensitivity to antifolates. Drug sensitivity analysis of WT, ∆Pf47GFP-luc-hdhfr and ∆Pf47GFP-luc to DHA ( D ), Chloroquine ( E ), Pyrimethamine ( F ), and WR99210 ( G ) in a dose dependent asexual growth inhibition assay using pLDH enzyme activity as a readout. Depicted data represents a triplicate measurement and was normalized to the MIN (10 μM DHA) and MAX (0.1% DMSO) controls. Error bars indicate standard deviations.

    Article Snippet: Pfs47 target regions, 5′hsp70 and 3′hsp86 fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen) sub-cloning.

    Techniques: Luciferase, Marker, Polymerase Chain Reaction, Chick Chorioallantoic Membrane Assay, Amplification, DNA Gel Electrophoresis, Selection, Growth Inhibition Assay, Activity Assay

    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Journal: PLoS ONE

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    doi: 10.1371/journal.pone.0115318

    Figure Lengend Snippet: A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Article Snippet: Phusion Hot Start DNA polymerases and GeneRuler 1 kb Plus DNA Ladder were purchased from Thermo Scientific (Waltham, MA).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated, Transformation Assay

    Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the Phusion HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of DNA bands in DNA marker (lane M) are indicated on the left.

    Journal: PLoS ONE

    Article Title: Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

    doi: 10.1371/journal.pone.0042341

    Figure Lengend Snippet: Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the Phusion HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of DNA bands in DNA marker (lane M) are indicated on the left.

    Article Snippet: For the cell viability screen in HEK293T cells, amplification of the barcode region for microarray analysis was performed using Phusion® Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific, Vantaa, Finland) and Decode negative selection primers.

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Transduction, shRNA, Real-time Polymerase Chain Reaction, Sequencing, Generated, Marker

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Article Snippet: Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct