phusion  (New England Biolabs)


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    Name:
    Phusion HF Buffer Pack
    Description:
    Phusion HF Buffer Pack 6 0 ml
    Catalog Number:
    B0518S
    Price:
    24
    Category:
    Buffers
    Size:
    6 0 ml
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    New England Biolabs phusion
    Phusion HF Buffer Pack
    Phusion HF Buffer Pack 6 0 ml
    https://www.bioz.com/result/phusion/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "In vitro synthesis of gene-length single-stranded DNA"

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24677-5

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Figure Legend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Techniques Used: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining

    2) Product Images from "Dissecting and Tuning Primer Editing by Proofreading Polymerases"

    Article Title: Dissecting and Tuning Primer Editing by Proofreading Polymerases

    Journal: bioRxiv

    doi: 10.1101/2021.05.11.443694

    Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase (n=3, error bars = +/-S.E.M.); B) NEB Q5 polymerase (n=3, error bars = +/-S.E.M.); C) Phusion polymerase (n=3, error bars = +/-S.E.M.).
    Figure Legend Snippet: Tuning of primer editing using phosphorothioate protection. Effect of incorporating phosphorothioate bonds into E. coli -specific 515F primers on extent of primer editing observed when the primer editing standards are amplified using A) KAPA HiFi polymerase (n=3, error bars = +/-S.E.M.); B) NEB Q5 polymerase (n=3, error bars = +/-S.E.M.); C) Phusion polymerase (n=3, error bars = +/-S.E.M.).

    Techniques Used: Amplification

    3) Product Images from "Solid-phase cloning for high-throughput assembly of single and multiple DNA parts"

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv036

    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
    Figure Legend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Techniques Used: Activity Assay, Selection, Construct

    4) Product Images from "In situ 10-cell RNA sequencing in tissue and tumor biopsy samples"

    Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-41235-9

    A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. ( A ) Poly(A) PCR was performed with 15 µg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). ( B ) Poly(A) PCR was performed with either 25, 5, 2.5, or 0.5 µg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analysed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analysed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times. Lanes were cropped by poly(A) PCR cycles for display but were electrophoresed on the same agarose gel and processed identically. The uncropped image is shown in Supplementary Fig. S13A .
    Figure Legend Snippet: A blend of Taq–Phusion polymerases improves selective poly(A) amplification of cDNA and reduces AL1 primer requirements. Cells were obtained by LCM from a human breast biopsy and split into 10-cell equivalent amplification replicates. ( A ) Poly(A) PCR was performed with 15 µg of AL1 primer with Taq alone (10 units), Phusion alone (4 units) or Taq/Phusion combination (3.75 units/1.5 units). ( B ) Poly(A) PCR was performed with either 25, 5, 2.5, or 0.5 µg of AL1 primer and the Taq–Phusion blend from (A). Above—Relative abundance for the indicated genes and preamplification conditions was measured by quantitative PCR (qPCR). Data are shown as the median inverse quantification cycle (40–Cq) ± range from n = 3 amplification replicates and were analysed by two-way (A) or one-way (B) ANOVA with replication. Below—Preamplifications were analysed by agarose gel electrophoresis to separate poly(A)-amplified cDNA from nonspecific, low molecular-weight concatemer (n.s.). Qualitatively similar results were obtained separately three times. Lanes were cropped by poly(A) PCR cycles for display but were electrophoresed on the same agarose gel and processed identically. The uncropped image is shown in Supplementary Fig. S13A .

    Techniques Used: Amplification, Laser Capture Microdissection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight

    5) Product Images from "RF-Cloning.org: an online tool for the design of restriction-free cloning projects"

    Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks396

    RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.
    Figure Legend Snippet: RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.

    Techniques Used: Clone Assay, Polyacrylamide Gel Electrophoresis, Generated, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Purification

    Related Articles

    Polymerase Chain Reaction:

    Article Title: RNA structure probing reveals the structural basis of Dicer binding and cleavage
    Article Snippet: .. To set up PCR reactions, 5 µL of RT products were mixed with 1 × Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB), 200 µM Solexa Forward and Reverse primers in a total volume of 50 µL. .. PCR was programmed as follows: stage I: 98 °C for 30 s; stage II: 98 °C for 10 s, 52 °C for 30 s, 72 °C for 15 s, repeating for total 22 cycles; Stage III: 72 °C for 5 min.

    Article Title: Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi
    Article Snippet: The PCR products were electrophoresed in 2% agarose gels, stained with ethidium bromide, and visualised under ultraviolet light. .. Cloning and DNA sequencing of T. evansi ITS and T. vivax TviCatL genes The PCR for T. evansi ITS, was performed in 20 μl of reaction mixture containing 4 μl of 5× Phusion® HF Buffer (1.5 mM MgCl2 was included in the final concentration), 1.6 μl of 200 μM dNTPs, 1 μl each of 1 μM IR1 and IR2 as a final concentration, 0.2 μl of Phusion® DNA polymerase (BioLabs, New England, USA) and 10.2 μl of sterile deionized distilled water. .. The entire ITS amplicon was gel-extracted using a QIAamp gel extraction kit (Qiagen), cloned and transformed to chemically-competent Escherichia coli (One Shot® Mach1™; ThermoFisher Scientific) using the TOPO® cloning procedure in accordance with the manufacturer’s instructions.

    Article Title: High-fidelity correction of genomic uracil by human mismatch repair activities
    Article Snippet: .. M13mp18 circular single-stranded (ss) (+) strand DNA (NEB) was annealed to PAGE purified oligonucleotide EDL330, 5'-CCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAG U TTGCATGCCTGCAGGTC, in a 1.4 ml reaction that contained 13.7 μg M13mp18 DNA, 80 pmol EDL330, 0.2 mM dNTPs, in Phusion polymerase HF buffer (NEB), to create a U•G mismatch at position 6283 (underlined); extended with Phusion polymerase (14 U) at 65°C for 45 min; and duplex extension products separated from other reaction components by two rounds of purification on PCR-pure spin columns (Qiagen, Valencia, CA). .. To ensure that no homoduplex molecules were present (which could in principle be produced by strand displacement, although this is not a reported activity of Phusion polymerase), products were digested with Hin dIII (1 U/μg substrate); single-stranded M13mp18 was removed by BND cellulose chromatography (Sigma, St Louis, MO); and linear molecules destroyed by treatment with Exonuclease V (USB, Cleveland, OH).

    Article Title: Pliocene-Early Pleistocene Geological Events Structure Pacific Martens (Martes caurina).
    Article Snippet: Marten mitochondrial baits were prepared using the protocol published by Maricic et al. (2010) with the following modifications: We used primer pairs mtI-F 5′-CAAGAGGAGAYAAGTCGTAACAAG-3′; mtI-R 5′-TCTCACCTATAATTTGACTTTGACA-3′; mtII-F 5′-AAGAAA GGAAGGAATCGAACC-3′; mtII-R 5′-TTGGAGTTGCACCAAT TTTTTG-3′; mtIII-F 5′-CATGGCTTTCTCAACTTTT-3′; mtIII-R 5′-CTTTGRTTTATCCAAGCACAC-3′ to obtain mitogenome fragments in 3 separate PCR reactions from individual OR-Cascade_ s48. .. Final concentrations of each PCR component were as follows: 0.5 µM each forward and reverse primer (desalted, Eurofins), 200 µM dNTPs (Thermo Scientific), 1× Phusion HF Buffer (New England Biolabs, hereafter written NEB), 0.006% DMSO (NEB), 1 Unit Phusion High-Fidelity DNA Polymerase (NEB), and 5-µl genomic DNA in a total reaction volume of 50 µl. .. Thermal cycling included a 98 °C initial denaturation for 1 min, 40 cycles at 98 °C for 10 s, annealing at 59 °C for 30 s, extension at 72 °C for 2 min, followed by a final extension at 72 °C for 5 min. Amplicons were extracted from a 0.7% agarose gel using a QIAGEN MinElute Gel Extraction kit with 2 final 10-µl elutions.

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: When using KOD Hot Start Master Mix, the following thermocycling setup was used: Initial denaturation at 95 °C for 2 min, followed by 20 step-down thermal cycles, each comprising denaturation at 95 °C for 20 s, annealing from 65 °C down to 55.5 °C for 10 s (0.5 °C decrement per cycle), and elongation at 70 °C for at the appropriate time (up to 135 s), then 5 thermal cycles with the constant annealing temperature (denaturation at 95 °C for 20 s, annealing at 62.5 °C for 10 s, elongation at 70 °C with the same time period as used in the step-down cycles), completed with a hold at 10 °C. .. For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB). .. Also, PCR thermocycling described above was modified in case of the ETR1 plasmids to have: (1) 11 step-down cycles with annealing starting from 65 °C down to 60 °C (i.e. decreasing by 0.5 °C in each subsequent cycle); (2) elongation time of at least 30 s per kbp of the target product (up to 200 s for the longest PCR products); and (3) final elongation time of 5 min.

    Hybridization:

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
    Article Snippet: .. Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization. .. Phusion HF was used as preferred buffer for extension unless the insert was PCR amplified using a different buffer; in those instances the matching buffer (e.g. Phusion GC buffer) was used also during the extension.

    Clone Assay:

    Article Title: Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi
    Article Snippet: The PCR products were electrophoresed in 2% agarose gels, stained with ethidium bromide, and visualised under ultraviolet light. .. Cloning and DNA sequencing of T. evansi ITS and T. vivax TviCatL genes The PCR for T. evansi ITS, was performed in 20 μl of reaction mixture containing 4 μl of 5× Phusion® HF Buffer (1.5 mM MgCl2 was included in the final concentration), 1.6 μl of 200 μM dNTPs, 1 μl each of 1 μM IR1 and IR2 as a final concentration, 0.2 μl of Phusion® DNA polymerase (BioLabs, New England, USA) and 10.2 μl of sterile deionized distilled water. .. The entire ITS amplicon was gel-extracted using a QIAamp gel extraction kit (Qiagen), cloned and transformed to chemically-competent Escherichia coli (One Shot® Mach1™; ThermoFisher Scientific) using the TOPO® cloning procedure in accordance with the manufacturer’s instructions.

    DNA Sequencing:

    Article Title: Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi
    Article Snippet: The PCR products were electrophoresed in 2% agarose gels, stained with ethidium bromide, and visualised under ultraviolet light. .. Cloning and DNA sequencing of T. evansi ITS and T. vivax TviCatL genes The PCR for T. evansi ITS, was performed in 20 μl of reaction mixture containing 4 μl of 5× Phusion® HF Buffer (1.5 mM MgCl2 was included in the final concentration), 1.6 μl of 200 μM dNTPs, 1 μl each of 1 μM IR1 and IR2 as a final concentration, 0.2 μl of Phusion® DNA polymerase (BioLabs, New England, USA) and 10.2 μl of sterile deionized distilled water. .. The entire ITS amplicon was gel-extracted using a QIAamp gel extraction kit (Qiagen), cloned and transformed to chemically-competent Escherichia coli (One Shot® Mach1™; ThermoFisher Scientific) using the TOPO® cloning procedure in accordance with the manufacturer’s instructions.

    Concentration Assay:

    Article Title: Trypanosoma vivax is the second leading cause of camel trypanosomosis in Sudan after Trypanosoma evansi
    Article Snippet: The PCR products were electrophoresed in 2% agarose gels, stained with ethidium bromide, and visualised under ultraviolet light. .. Cloning and DNA sequencing of T. evansi ITS and T. vivax TviCatL genes The PCR for T. evansi ITS, was performed in 20 μl of reaction mixture containing 4 μl of 5× Phusion® HF Buffer (1.5 mM MgCl2 was included in the final concentration), 1.6 μl of 200 μM dNTPs, 1 μl each of 1 μM IR1 and IR2 as a final concentration, 0.2 μl of Phusion® DNA polymerase (BioLabs, New England, USA) and 10.2 μl of sterile deionized distilled water. .. The entire ITS amplicon was gel-extracted using a QIAamp gel extraction kit (Qiagen), cloned and transformed to chemically-competent Escherichia coli (One Shot® Mach1™; ThermoFisher Scientific) using the TOPO® cloning procedure in accordance with the manufacturer’s instructions.

    Polyacrylamide Gel Electrophoresis:

    Article Title: High-fidelity correction of genomic uracil by human mismatch repair activities
    Article Snippet: .. M13mp18 circular single-stranded (ss) (+) strand DNA (NEB) was annealed to PAGE purified oligonucleotide EDL330, 5'-CCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAG U TTGCATGCCTGCAGGTC, in a 1.4 ml reaction that contained 13.7 μg M13mp18 DNA, 80 pmol EDL330, 0.2 mM dNTPs, in Phusion polymerase HF buffer (NEB), to create a U•G mismatch at position 6283 (underlined); extended with Phusion polymerase (14 U) at 65°C for 45 min; and duplex extension products separated from other reaction components by two rounds of purification on PCR-pure spin columns (Qiagen, Valencia, CA). .. To ensure that no homoduplex molecules were present (which could in principle be produced by strand displacement, although this is not a reported activity of Phusion polymerase), products were digested with Hin dIII (1 U/μg substrate); single-stranded M13mp18 was removed by BND cellulose chromatography (Sigma, St Louis, MO); and linear molecules destroyed by treatment with Exonuclease V (USB, Cleveland, OH).

    Purification:

    Article Title: High-fidelity correction of genomic uracil by human mismatch repair activities
    Article Snippet: .. M13mp18 circular single-stranded (ss) (+) strand DNA (NEB) was annealed to PAGE purified oligonucleotide EDL330, 5'-CCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAG U TTGCATGCCTGCAGGTC, in a 1.4 ml reaction that contained 13.7 μg M13mp18 DNA, 80 pmol EDL330, 0.2 mM dNTPs, in Phusion polymerase HF buffer (NEB), to create a U•G mismatch at position 6283 (underlined); extended with Phusion polymerase (14 U) at 65°C for 45 min; and duplex extension products separated from other reaction components by two rounds of purification on PCR-pure spin columns (Qiagen, Valencia, CA). .. To ensure that no homoduplex molecules were present (which could in principle be produced by strand displacement, although this is not a reported activity of Phusion polymerase), products were digested with Hin dIII (1 U/μg substrate); single-stranded M13mp18 was removed by BND cellulose chromatography (Sigma, St Louis, MO); and linear molecules destroyed by treatment with Exonuclease V (USB, Cleveland, OH).

    Article Title: EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains
    Article Snippet: All the marker gene cassettes (Fig. , Supplementary material Table S2) were synthesized by GeneArt (Life Technologies). .. DNA fragments were gel purified and incubated in HF buffer (New England BioLabs) together with USER enzyme (New England BioLabs) for 25 min at 37 °C followed by incubation at 25 °C for 25 min. .. The reactions were transformed into chemically competent E. coli cells.

    Incubation:

    Article Title: EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains
    Article Snippet: All the marker gene cassettes (Fig. , Supplementary material Table S2) were synthesized by GeneArt (Life Technologies). .. DNA fragments were gel purified and incubated in HF buffer (New England BioLabs) together with USER enzyme (New England BioLabs) for 25 min at 37 °C followed by incubation at 25 °C for 25 min. .. The reactions were transformed into chemically competent E. coli cells.

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    New England Biolabs phusion high fidelity dna polymerase
    Cloned pre-mir-122 stem-loop region sequences from HepG2 <t>DNA</t> show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with <t>Phusion</t> high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/New England Biolabs
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    New England Biolabs phusion buffer
    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, <t>Phusion</t> and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.
    Phusion Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion buffer/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    86
    New England Biolabs phusion
    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. <t>Phusion;</t> 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.
    Phusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion - by Bioz Stars, 2021-07
    86/100 stars
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    Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Journal: PLoS ONE

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells

    doi: 10.1371/journal.pone.0122471

    Figure Lengend Snippet: Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes. (A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10 -7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T) n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.

    Article Snippet: The observed error rate was ~14-fold higher than that reported for Phusion High-Fidelity DNA Polymerase (GC Buffer) (New England Biolabs; ) which, if this error rate is correct, suggests that these changes are unlikely to have arisen solely as a result of PCR errors.

    Techniques: Clone Assay, Amplification, Sequencing, Polymerase Chain Reaction, Variant Assay

    Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Journal: Scientific Reports

    Article Title: TT(N)mGCCTC inhibits archaeal family B DNA polymerases

    doi: 10.1038/s41598-018-20127-4

    Figure Lengend Snippet: Olig2.5 inhibited archaeal family B DNA polymerases. An E. coli DH5α colony harbouring the 2.6 kb upstream sequence of Olig2 in pGL3-Basic vector was cultured and 1 μl of the culture was directly used as template for PCR. Olig2F and OligTSSR were used as primers to amplify the 2 kb fragment. Olig2.5 was added to see its inhibition to various DNAP. DNAP: DNA polymerase. LA: LA Taq DNAP. Q5: Q5 High-Fidelity DNAP. PS: PrimeSTAR HS DNAP. GXL: PrimeSTAR GXL DNAP. Phusion: Phusion High-Fidelity DNAP. Cobuddy: Cobuddy Super Fidelity DNAP. KOD: KOD DNAP.

    Article Snippet: Phusion® High-Fidelity DNA Polymerase (Phusion) and Q5® High-Fidelity DNA Polymerase (Q5) were from New England Biolabs.

    Techniques: Sequencing, Plasmid Preparation, Cell Culture, Polymerase Chain Reaction, Inhibition

    Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Journal: Nucleic Acids Research

    Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

    doi: 10.1093/nar/gkv036

    Figure Lengend Snippet: Results from head-to-tail SPC. ( A ) Comparison of compatibility of thermostable polymerases with the head-to-tail method. The polymerases with best proof-reading capabilities, Phusion and Deep Vent, all failed to generate transformants with the standard head-to-tail protocol. ( B ) Activity was retained for protocols using Phusion after supplementation of the washing buffer with SDS. ( C ) Colony screens of inserted region representing a selection of assemblies of various lengths and number of inserts assembled by head-to-tail SPC. Final construct sizes spanned 2.9 to 7.2 kbps.

    Article Snippet: Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization.

    Techniques: Activity Assay, Selection, Construct

    ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Journal: Scientific Reports

    Article Title: In vitro synthesis of gene-length single-stranded DNA

    doi: 10.1038/s41598-018-24677-5

    Figure Lengend Snippet: ssDNA production by aPCR. ( a ) aPCR reactions were assembled with a 50-molar excess of a forward primer for the amplification of a 1,000 nt ssDNA fragment using the M13mp18 ssDNA plasmid as template, and with 10 different polymerases that were tested for highest yield of ssDNA production (upper band: expected dsDNA size is 1,000 bp; lower band: expected ssDNA size is 1,000 nt) as judged by agarose gel electrophoresis (right panel). QuantaBio AccuStart HiFi, polymerase (lane 2, boxed) produced the highest amount without overlapping dsDNA contaminants. 1. Accustart; 2. Accustart HiFi; 3. Accustart II; 4. AccuPrime; 5. GoTaq; 6. DreamTaq; 7. Phusion; 8. Platinum SuperFi; 9. Q5; 10. Tth polymerase. ( b ) Biochemical validation of ssDNA production by incubating 1,000 nt aPCR reaction products with the ssDNA-specific ExoI or S1 nucleases or dsDNA-specific restriction enzymes Eco RI and Nae I (left panel). Agarose gel electrophoresis of the digestion products as labeled by lane (right panel). M: Marker, C: aPCR product control, ExoI: exonuclease I, S1: S1 nuclease, Enz: Eco RI + Nae I. ( c ) NEB LongAmp was used to generate ssDNA up to 15,000 nt long using lambda phage dsDNA as template. Purification of the 10 kb fragment shows a single band of higher molecular weight than the M13mp18 ssDNA (7,249 nt). ( d ) The primer design algorithm aPrime was used to select primers for product sizes between 500 and 3,000 nt using M13mp18 ssDNA as template and the Quantabio Accustart HiFi enzyme. SYBR Safe stained agarose gels illuminated under blue light show dsDNA as yellow bands, while ssDNA show as orange bands.

    Article Snippet: The following enzymes were purchased from the respective commercial providers to test enzymatic production of ssDNA: AccuStart™, AccuStart™ II, and AccuStart™ HiFi from Quantabio; Q5® hot start HiFi, Phusion®, LongAmp®, Deep Vent®, and Deep Vent® (exo-) from New England BioLabs Inc. (NEB); AccuPrime™, Platinum™ SuperFi™, Tth and DreamTaq™ from ThermoFisher Scientific Inc.; GoTaq® from Promega (Promega corp.); and LA Taq ® from Takara Bio.

    Techniques: Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Produced, Labeling, Marker, Purification, Molecular Weight, Staining