phusion  (New England Biolabs)


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  • 99
    Name:
    Phusion HF Buffer Pack
    Description:

    Catalog Number:
    B0518S
    Price:
    None
    Score:
    85
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    Structured Review

    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.

    https://www.bioz.com/result/phusion/product/New England Biolabs
    Average 99 stars, based on 105 article reviews
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    phusion - by Bioz Stars, 2019-10
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    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 "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

    3) 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

    4) 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

    Clone Assay:

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. At the end of the first round of asymmetric PCR, the two reactions were combined and allowed to proceed through 20 more PCR amplification cycles.

    Article Title: Identification of multiple integrin ?1 homologs in zebrafish (Danio rerio)
    Article Snippet: PCR reactions were performed using Phusion (New England Biolabs). .. The PCR reactions generated products of ~2.4 kB for β1–1, β1–2 and β1–3, and ~1.9 kB for β1tr-1, β1tr-2 and β1tr-3.

    Article Title: Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769
    Article Snippet: Extraction of bacterial genomic DNA was carried out using cell lysis under heating in the presence of sodium dodecyl sulfate (SDS) and proteinase K, followed by purification with a phenol:chloroform:isoamylic alcohol solution (PCI) and subsequent dialysis in TE buffer pH 8.0. .. The cloning of the genes related to the production of bacterial cellulose was carried out with amplification reactions (PCR) using specifically designed primers in the presence of Pfx Plantinum (Invitrogen) or Phusion (New England Biolabs) polymerases. .. The cloned genes were ligated to pTZ57R/T vector, transformed into ultracompetent Escherichia coli (DH10B) and sequencing was carried out using Big Dye ® Terminator v3.1 Cycle Sequencing Kit and the Genetic Analyzer 3130 (Applied Biosystems).

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Constructs were created using the Gateway technology as recommended by the manufacturer (Life Technologies, Carlsbad, CA). ripTPS was cloned from the genomic DNA of R. solanacearum GMI1000 in pDONR207, creating pMP12 (with a stop codon) and pMP15 (without a stop codon). .. Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. The three mutant alleles of ripTPS were generated using the QuikChange XL site-directed mutagenesis kit (Agilent) on the pMP15 plasmid, creating plasmids pMP90 (Y154V), pMP91 (W163S), and pMP92 (D208G), respectively.

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: All PCR products for cloning purposes were amplified in 35 cycles using proof-reading PfuX7 polymerase [ ], by touch-down PCR programs with maximum annealing temperature interval ranging from 68–59°C or 64–57°C. .. Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: Paragraph title: 4. Cloning of aldo/keto reductase in E . coli BL21 ... The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Centrifugation:

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs). .. The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs).

    Amplification:

    Article Title: Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo
    Article Snippet: Transposed DNA was purified using Qiagen Minelute kit. .. Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011). .. Libraries were then purified with Ampure Beads at a 1.2: 1 beads to sample ratio and sequenced on the Hiseq4000 using 100bp paired end reads.

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Further, the third hypervariable (V3) region of 16S ribosomal RNA gene (approx 280 bp) was amplified (PCR1) from 10 ng genomic DNA using specific primers 341F 5′CCTACGGGAGGCAGCAG3′ and 518R 5′ATTACCGCGGCTGCTGG3′ in a PCR thermocycler. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Tagmentation was performed in a final volume of 5 μl using 5 ng of purified PCR product, 0.15 μl of Nextera tagment enzyme and tagmentation buffer previously described by Wang et al . .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs). .. PCR cycling conditions used were as follows: 72 °C for 3 minutes, 98 °C for 2 minutes and 15 cycles of 98 °C for 10 seconds, 63 °C for 30 secconds, and 72 °C for 3 minutes.

    Article Title: Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation
    Article Snippet: Paragraph title: Single-genome Nested Amplification of SIVmac251 env ... The following master mix was made at room temperature: 5 ul of 5# Phusion HF buffer (New England BioLabs, Ipswich, MA), 0.5 ul of 10 mM dNTPs, 0.75 ul of 10 uM 251envF1 (5%-CAG TCT TTT ATG GTG TAC CAG CTT GGA GGA ATG-3%), 0.75 ul of 10 uM 251envR1 (5%-GAG GAT CCA TCT TCC ACC TCT CCT AAG AGT C-3%), 0.2 ul of Phusion Hot Start high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and 14.8 ul of double-distilled water (ddH2O).

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. In a separate tube a similar reaction was performed using mutant specific primers EBS1delta (100 μM) and EBS2 (10 μM) primers.

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: A new product strand does not contain a binding site for the reverse megaprimer and is therefore not a template for the next round of PCR, causing a linear rather than exponential amplification. .. The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB).

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: The reaction mixture was neutralized by adding 20 μL of 1 N HCl. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix. .. The amplification primers used were 5′ -AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′ and 5′ -CAAGCAGAAGACGGCATACGA-3′ .

    Article Title: Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene
    Article Snippet: PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb. .. PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb.

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: The products were amplified in a 50 µl reaction, the final concentrations of 100 F/R and 300 F/R were 0.133 µM, 200 F/R were 0.200 µM, and 400 F/R were 0.067 µM. .. PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA).

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: Following further extensive experimentation with primers designed against the S. italica sequence, we used the following protocols to amplify the region corresponding to the entire mature peptide for the S and L loci in the eight plants previously analyzed for starch protein and enzyme activity. .. The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs). .. Cycling conditions were 30 s at 98 °C; 40 cycles of 10 s at 98 °C, and 2 min 30 s at 72 °C; final extension step of 10 min at 72 °C.

    Article Title: Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769
    Article Snippet: Extraction of bacterial genomic DNA was carried out using cell lysis under heating in the presence of sodium dodecyl sulfate (SDS) and proteinase K, followed by purification with a phenol:chloroform:isoamylic alcohol solution (PCI) and subsequent dialysis in TE buffer pH 8.0. .. The cloning of the genes related to the production of bacterial cellulose was carried out with amplification reactions (PCR) using specifically designed primers in the presence of Pfx Plantinum (Invitrogen) or Phusion (New England Biolabs) polymerases. .. The cloned genes were ligated to pTZ57R/T vector, transformed into ultracompetent Escherichia coli (DH10B) and sequencing was carried out using Big Dye ® Terminator v3.1 Cycle Sequencing Kit and the Genetic Analyzer 3130 (Applied Biosystems).

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: All PCR products for cloning purposes were amplified in 35 cycles using proof-reading PfuX7 polymerase [ ], by touch-down PCR programs with maximum annealing temperature interval ranging from 68–59°C or 64–57°C. .. Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The open reading frame all2316 , encoding aldo/keto reductase was amplified by polymerase chain reaction using genomic DNA as the template. .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Synthesized:

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: To ligate 5′ small RNA adaptor (5′ -GUUCAGAGUUCUACAGUCCGACGAUC-3′ ) to the 5′ -end of the mono-phosphorylated RNA, the enriched RNA samples were incubated with 100 μM of the adaptor and 2.5 U of T4 RNA ligase (New England BioLabs, Ipswich, MA). cDNAs were synthesized using the adaptor-ligated mRNAs as template using a modified small RNA reverse transcriptase (RT) primer from Illumina (5′ -CAAGCAGAAGACGGCATACGANNNNNNNNN-3′ ) and Superscript II Reverse Transcriptase (Life Technologies, Carlsbad, CA). .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix.

    Picogreen Assay:

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Amplicons were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) following the manufacture’s recommendations. .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs).

    Construct:

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Paragraph title: DNA manipulations and genetic constructs. ... Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA).

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO. .. All vectors were assembled by USER cloning or USER fusion as described previously [ , ].

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The purified PCR product was digested with Nde1 and Xho1 (NEB), and the resultant DNA fragment was cloned into the pET-21a expression vector (Novagen), digested with the same restriction enzymes. pET21a incorporates a C-terminal histidine (6x His) tag to aid purification.

    Real-time Polymerase Chain Reaction:

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl. .. To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl.

    IA:

    Article Title: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response
    Article Snippet: Sequences were designed using DNAWorks [ ] and purchased from Integrated DNA Technologies, Inc. (Coralville, IA). .. A 10μL volume of outer primer mix was added to 12.7μL of inner primer mix, along with 1μL of 10mM DNTPs, 6μL of 5x Phusion buffer, and 0.3μL of Phusion polymerase (NEB, Waltham, MA) for a final volume of 30μL.

    Incubation:

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: Reverse transcription was carried out at 25°C for 10 min, 37°C for 60 min, and 42°C for 60 min, followed by incubation at 70°C for 10 min. After the reaction, RNA was hydrolyzed by adding 20 μL of 1 N NaOH and incubation at 65°C for 30 min. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix.

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Pre-selection libraries were also separately incubated with 2 U of BspMI restriction endonuclease (NEB) in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.9) for 1 h at 37 °C. .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: Paragraph title: Multiplex PCR FFPE DNA Quality Assessment ... PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA).

    Activity Assay:

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: Following further extensive experimentation with primers designed against the S. italica sequence, we used the following protocols to amplify the region corresponding to the entire mature peptide for the S and L loci in the eight plants previously analyzed for starch protein and enzyme activity. .. The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs).

    Expressing:

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA).

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Modification:

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: The RNA was mixed with 25 μM modified small RNA RT primer and incubated at 70°C for 10 min and then at 25°C for 10 min. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix.

    Over Expression:

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: For cloning and over-expression analysis, genomic DNA from Anabaena 7120 was extracted following the protocol of Srivastava et al. [ ]. .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Nested PCR:

    Article Title: Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation
    Article Snippet: A 5-fold dilution series was made from cDNA in TE buffer (10 mM Tris [pH 8.0], 0.1 mM EDTA; Integrated DNA Technologies), and nested PCR was performed. .. The following master mix was made at room temperature: 5 ul of 5# Phusion HF buffer (New England BioLabs, Ipswich, MA), 0.5 ul of 10 mM dNTPs, 0.75 ul of 10 uM 251envF1 (5%-CAG TCT TTT ATG GTG TAC CAG CTT GGA GGA ATG-3%), 0.75 ul of 10 uM 251envR1 (5%-GAG GAT CCA TCT TCC ACC TCT CCT AAG AGT C-3%), 0.2 ul of Phusion Hot Start high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and 14.8 ul of double-distilled water (ddH2O).

    Derivative Assay:

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: The sequence of Bacillus anthracis genomic DNA to be targeted for disruption was scanned for potential intron insertion sites using a computer algorithm derived from a learning set of successful intron integrations [ ]. .. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template.

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. Expression plasmids (pMP72, pMP93, pMP94, and pMP95) were created by LR Gateway cloning using pAM-PAT-P35S-GW-3xHA (L. D. Noël and J. E. Parker, unpublished data) and used to transform Agrobacterium tumefaciens GV3101::pMP90RK.

    Hybridization:

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases. .. The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases.

    Flow Cytometry:

    Article Title: Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo
    Article Snippet: Transposed DNA was purified using Qiagen Minelute kit. .. Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011). .. Libraries were then purified with Ampure Beads at a 1.2: 1 beads to sample ratio and sequenced on the Hiseq4000 using 100bp paired end reads.

    Ligation:

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: Ligation products were purified by the Agencourt AMPureXP system (Beckman Coulter, Brea, CA), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor dsDNA System (Promega). .. To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The purified PCR product was digested with Nde1 and Xho1 (NEB), and the resultant DNA fragment was cloned into the pET-21a expression vector (Novagen), digested with the same restriction enzymes. pET21a incorporates a C-terminal histidine (6x His) tag to aid purification.

    Generated:

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Illumina sequencing libraries were generated using the Nextera DNA Library Prep Kit (Illumina) following the manufacture’s recommendations with the following changes. .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs).

    Article Title: Identification of multiple integrin ?1 homologs in zebrafish (Danio rerio)
    Article Snippet: PCR reactions were performed using Phusion (New England Biolabs). .. Cycling parameters were 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 60°C for 20 s and 72°C for 60 s. An additional sequence, β1tr-3, was amplified from reverse transcribed RNA from adult kidney (prepared as described below) using the same primers as for β1tr-2.

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. Expression plasmids (pMP72, pMP93, pMP94, and pMP95) were created by LR Gateway cloning using pAM-PAT-P35S-GW-3xHA (L. D. Noël and J. E. Parker, unpublished data) and used to transform Agrobacterium tumefaciens GV3101::pMP90RK.

    Gel Extraction:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration. .. PCR reaction was set at 98 °C for 30 s, 30 cycles of 98 °C for 10 s, 72 °C for 30 s, extension at 72 °C for 5 s. PCR products were run on 2% agarose gel with SYBR Safe DNA gel stain.

    Article Title: Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene
    Article Snippet: PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb. .. Once sex-specific size differences were discovered, primers were designed to fully amplify across the region, with amplification and sequencing performed following Genome Walking PCR protocol, with 2 min extensions.

    Polymerase Chain Reaction:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Further, the third hypervariable (V3) region of 16S ribosomal RNA gene (approx 280 bp) was amplified (PCR1) from 10 ng genomic DNA using specific primers 341F 5′CCTACGGGAGGCAGCAG3′ and 518R 5′ATTACCGCGGCTGCTGG3′ in a PCR thermocycler. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Tagmentation was performed in a final volume of 5 μl using 5 ng of purified PCR product, 0.15 μl of Nextera tagment enzyme and tagmentation buffer previously described by Wang et al . .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs). .. PCR cycling conditions used were as follows: 72 °C for 3 minutes, 98 °C for 2 minutes and 15 cycles of 98 °C for 10 seconds, 63 °C for 30 secconds, and 72 °C for 3 minutes.

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: Ligation products were purified by the Agencourt AMPureXP system (Beckman Coulter, Brea, CA), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor dsDNA System (Promega). .. To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl. .. PCR was performed under the following temperature profiles: 30 s at 94°C, 15 cycles of 10 s at 94°C, 30 s at 60°C, 1 min at 72°C, a final extension step of 5 min at 72°C, and storage at 4°C.

    Article Title: Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation
    Article Snippet: The following master mix was made at room temperature: 5 ul of 5# Phusion HF buffer (New England BioLabs, Ipswich, MA), 0.5 ul of 10 mM dNTPs, 0.75 ul of 10 uM 251envF1 (5%-CAG TCT TTT ATG GTG TAC CAG CTT GGA GGA ATG-3%), 0.75 ul of 10 uM 251envR1 (5%-GAG GAT CCA TCT TCC ACC TCT CCT AAG AGT C-3%), 0.2 ul of Phusion Hot Start high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and 14.8 ul of double-distilled water (ddH2O). .. A 23 ul volume of master mix and 2 ul of cDNA or DNA from 5-fold serial dilutions in TE were added to 0.2-ml tubes.

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: The resulting insertion sites are shown in Table and the mutations necessary to retarget the intron to a desired site were introduced through PCR mutagenesis using two pairs of partially overlapping primers (Table ) to assemble a short DNA fragment flanked by HindIII and BsrG1 sites and incorporating the changes within the intron required to redirect the intron to a new locus as described previously [ ]. .. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. In a separate tube a similar reaction was performed using mutant specific primers EBS1delta (100 μM) and EBS2 (10 μM) primers.

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: A new product strand does not contain a binding site for the reverse megaprimer and is therefore not a template for the next round of PCR, causing a linear rather than exponential amplification. .. The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB). .. The amount of megaprimer has to be screened.

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: The reaction mixture was neutralized by adding 20 μL of 1 N HCl. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix. .. The amplification primers used were 5′ -AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′ and 5′ -CAAGCAGAAGACGGCATACGA-3′ .

    Article Title: Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene
    Article Snippet: PCR primers were designed to sequences containing sex-linked polymorphisms, as well as genes predicted to be adjacent and between these markers based on the stickleback BLAT. .. PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb.

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: Indexing adapters were provided by the Broad Institute (Cambridge, MA). .. The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases. .. DNA purification was done using QIAquick and MinElute PCR purification kits (Qiagen; Valencia, CA).

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: Multiplex PCR reaction conditions were as follows: 98°C for 30 sec, followed by 35 cycles each of 98°C for 10 sec, 62°C for 30 sec, and 72°C for 30 sec with a final 10 min extension at 72°C. .. PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA). .. For positive controls, the same PCR reaction was performed on corresponding normal samples, as well as on a reference prostate gDNA, R1.

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Blunt-ended post-selection library members or sticky-ended pre-selection library members were purified with the QIAQuick PCR Purification Kit (Qiagen) and ligated to 10 pmol adapter1/2(AACA) (Cas9:v2.1 sgRNA, 100 nM), adapter1/2(TTCA) (Cas9:v2.1 sgRNA, 1000 nM), adapter1/2 (Cas9:v2.1 sgRNA, 1000 nM), or lib adapter1/CLTA(#) lib adapter 2 (pre-selection) with 1,000 U of T4 DNA Ligase (NEB) in NEB T4 DNA Ligase Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM magnesium chloride, 1 mM ATP, 10 mM dithiothreitol) overnight ( > 10 h) at room temperature. .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: Identification of multiple integrin ?1 homologs in zebrafish (Danio rerio)
    Article Snippet: The following primers were used: β1–1, 5'-ATGGACCTGAAGCTACTTTTCATATC-3' and 5'-CTGATGGCCATTATTTGCCTTCG-3', β1–2, 5'-ATGGACGTAAGGCTGCTCCTG-3'and 5'-CACGTTCGTCCATTATTTGCCCTC-3', β1–3, 5'-ATGAAAATGAAGCTGCTGTTATTATC-3' and 5'-CACTTTCCCTCATATCTGGGATTC-3' β1tr-1, 5'-ATGGATATAACAGTTTTGTTATTATCAG-3' and 5'-ATGTATAACATGAGGTCATGATGTAC-3' β1tr-2 5'-ATGGATATAACAGTTTTGTTATTATCAG-3' and 5'-GTATAACATGTGTCTCAATATATGATG-3' Total RNA was prepared from 4-day old embryos using TRI reagent (Sigma), and reverse transcription was performed using Superscript II (Invitrogen) according to the manufacturer's instructions. .. PCR reactions were performed using Phusion (New England Biolabs). .. Cycling parameters were 98°C for 30 s, followed by 40 cycles of 98°C for 10 s, 60°C for 20 s and 72°C for 60 s. An additional sequence, β1tr-3, was amplified from reverse transcribed RNA from adult kidney (prepared as described below) using the same primers as for β1tr-2.

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: Following further extensive experimentation with primers designed against the S. italica sequence, we used the following protocols to amplify the region corresponding to the entire mature peptide for the S and L loci in the eight plants previously analyzed for starch protein and enzyme activity. .. The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs). .. Cycling conditions were 30 s at 98 °C; 40 cycles of 10 s at 98 °C, and 2 min 30 s at 72 °C; final extension step of 10 min at 72 °C.

    Article Title: Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769
    Article Snippet: Extraction of bacterial genomic DNA was carried out using cell lysis under heating in the presence of sodium dodecyl sulfate (SDS) and proteinase K, followed by purification with a phenol:chloroform:isoamylic alcohol solution (PCI) and subsequent dialysis in TE buffer pH 8.0. .. The cloning of the genes related to the production of bacterial cellulose was carried out with amplification reactions (PCR) using specifically designed primers in the presence of Pfx Plantinum (Invitrogen) or Phusion (New England Biolabs) polymerases. .. The cloned genes were ligated to pTZ57R/T vector, transformed into ultracompetent Escherichia coli (DH10B) and sequencing was carried out using Big Dye ® Terminator v3.1 Cycle Sequencing Kit and the Genetic Analyzer 3130 (Applied Biosystems).

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Constructs were created using the Gateway technology as recommended by the manufacturer (Life Technologies, Carlsbad, CA). ripTPS was cloned from the genomic DNA of R. solanacearum GMI1000 in pDONR207, creating pMP12 (with a stop codon) and pMP15 (without a stop codon). .. Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. The three mutant alleles of ripTPS were generated using the QuikChange XL site-directed mutagenesis kit (Agilent) on the pMP15 plasmid, creating plasmids pMP90 (Y154V), pMP91 (W163S), and pMP92 (D208G), respectively.

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: Paragraph title: PCR, vector construction and protospacers ... Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Article Title: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response
    Article Snippet: Paragraph title: Recursive PCR assembly ... A 10μL volume of outer primer mix was added to 12.7μL of inner primer mix, along with 1μL of 10mM DNTPs, 6μL of 5x Phusion buffer, and 0.3μL of Phusion polymerase (NEB, Waltham, MA) for a final volume of 30μL.

    Sonication:

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: DNA samples were fragmented by sonication (Covaris; Woburn, MA) and the fragments end-repaired, A-tailed, and ligated to indexing oligonucleotide adapters using NEBNext reagents (New England Biolabs; Ipswich, MA). .. The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases.

    Binding Assay:

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: A new product strand does not contain a binding site for the reverse megaprimer and is therefore not a template for the next round of PCR, causing a linear rather than exponential amplification. .. The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB).

    DNA Extraction:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Paragraph title: Midgut isolation, DNA extraction, 16S rRNA gene sequencing, and analysis ... The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: Paragraph title: DNA Extraction, Amplification, and Analysis ... The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs).

    Nucleic Acid Electrophoresis:

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA). .. All PCR reactions were purified using the MinElute PCR Cleanup Kit (QIAGEN, Venlo, Netherlands), and quantified with a Qubit 1.0 fluorimeter (Invitrogen).

    Mutagenesis:

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: The resulting insertion sites are shown in Table and the mutations necessary to retarget the intron to a desired site were introduced through PCR mutagenesis using two pairs of partially overlapping primers (Table ) to assemble a short DNA fragment flanked by HindIII and BsrG1 sites and incorporating the changes within the intron required to redirect the intron to a new locus as described previously [ ]. .. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. In a separate tube a similar reaction was performed using mutant specific primers EBS1delta (100 μM) and EBS2 (10 μM) primers.

    Isolation:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Paragraph title: Midgut isolation, DNA extraction, 16S rRNA gene sequencing, and analysis ... The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. After ligation the construct pET-21a –AKR and empty vector pET-21a were introduced into E . coli strain BL21 (DE3) for expression studies.

    Size-exclusion Chromatography:

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: Multiplex PCR reaction conditions were as follows: 98°C for 30 sec, followed by 35 cycles each of 98°C for 10 sec, 62°C for 30 sec, and 72°C for 30 sec with a final 10 min extension at 72°C. .. PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA).

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Purification:

    Article Title: Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo
    Article Snippet: Transposed DNA was purified using Qiagen Minelute kit. .. Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011).

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration. .. PCR reaction was set at 98 °C for 30 s, 30 cycles of 98 °C for 10 s, 72 °C for 30 s, extension at 72 °C for 5 s. PCR products were run on 2% agarose gel with SYBR Safe DNA gel stain.

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Tagmentation was performed in a final volume of 5 μl using 5 ng of purified PCR product, 0.15 μl of Nextera tagment enzyme and tagmentation buffer previously described by Wang et al . .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs).

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: Ligation products were purified by the Agencourt AMPureXP system (Beckman Coulter, Brea, CA), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor dsDNA System (Promega). .. To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl.

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. At the end of the first round of asymmetric PCR, the two reactions were combined and allowed to proceed through 20 more PCR amplification cycles.

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB). .. The 2nd RF PCR reaction starts with an initial denaturation step (30 s, 98°C), followed by 35 cycles of denaturation (10 s, 98°C), annealing (30s, Tm (F1/R1) +3°C) and extension (30 s/1 kb, 72°C) with no final extension.

    Article Title: Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene
    Article Snippet: PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb. .. Once sex-specific size differences were discovered, primers were designed to fully amplify across the region, with amplification and sequencing performed following Genome Walking PCR protocol, with 2 min extensions.

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases. .. DNA purification was done using QIAquick and MinElute PCR purification kits (Qiagen; Valencia, CA).

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA). .. PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA).

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Blunt-ended post-selection library members or sticky-ended pre-selection library members were purified with the QIAQuick PCR Purification Kit (Qiagen) and ligated to 10 pmol adapter1/2(AACA) (Cas9:v2.1 sgRNA, 100 nM), adapter1/2(TTCA) (Cas9:v2.1 sgRNA, 1000 nM), adapter1/2 (Cas9:v2.1 sgRNA, 1000 nM), or lib adapter1/CLTA(#) lib adapter 2 (pre-selection) with 1,000 U of T4 DNA Ligase (NEB) in NEB T4 DNA Ligase Reaction Buffer (50 mM Tris-HCl, pH 7.5, 10 mM magnesium chloride, 1 mM ATP, 10 mM dithiothreitol) overnight ( > 10 h) at room temperature. .. Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection). .. Amplified DNAs were gel purified, quantified with the KAPA Library Quantification Kit-Illumina (KAPA Biosystems) and subjected to single-read sequencing on an Illumina MiSeq or Rapid Run single-read sequencing on an Illumina HiSeq 2500 (Harvard University FAS Center for Systems Biology Core facility, Cambridge, MA).

    Article Title: Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769
    Article Snippet: Extraction of bacterial genomic DNA was carried out using cell lysis under heating in the presence of sodium dodecyl sulfate (SDS) and proteinase K, followed by purification with a phenol:chloroform:isoamylic alcohol solution (PCI) and subsequent dialysis in TE buffer pH 8.0. .. The cloning of the genes related to the production of bacterial cellulose was carried out with amplification reactions (PCR) using specifically designed primers in the presence of Pfx Plantinum (Invitrogen) or Phusion (New England Biolabs) polymerases.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The amplified product was gel purified using a QIAquick gel extraction kit (Qiagen).

    Article Title: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response
    Article Snippet: A 10μL volume of outer primer mix was added to 12.7μL of inner primer mix, along with 1μL of 10mM DNTPs, 6μL of 5x Phusion buffer, and 0.3μL of Phusion polymerase (NEB, Waltham, MA) for a final volume of 30μL. .. A 1.25μL aliquot of the first PCR reaction product was added to 5μL of 5x Phusion Buffer, 0.75μL 10mM DNTPs, 2μL of outer primer mix, and 0.25μL of Phusion polymerase in 25μL.

    Sequencing:

    Article Title: Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo
    Article Snippet: Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011). .. Libraries were then purified with Ampure Beads at a 1.2: 1 beads to sample ratio and sequenced on the Hiseq4000 using 100bp paired end reads.

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Paragraph title: Midgut isolation, DNA extraction, 16S rRNA gene sequencing, and analysis ... The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Illumina sequencing libraries were generated using the Nextera DNA Library Prep Kit (Illumina) following the manufacture’s recommendations with the following changes. .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs).

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: The sequence of Bacillus anthracis genomic DNA to be targeted for disruption was scanned for potential intron insertion sites using a computer algorithm derived from a learning set of successful intron integrations [ ]. .. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template.

    Article Title: Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene
    Article Snippet: PCR primers were designed to sequences containing sex-linked polymorphisms, as well as genes predicted to be adjacent and between these markers based on the stickleback BLAT. .. PCRs were performed to amplify the sequence between the markers and genes, looking for sex-specific amplicons; long PCRs were performed following the Phusion (NEB) protocol with 35 cycles and 15 s/kb. .. Once sex-specific size differences were discovered, primers were designed to fully amplify across the region, with amplification and sequencing performed following Genome Walking PCR protocol, with 2 min extensions.

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: Paragraph title: Targeted sequence capture ... The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases.

    Article Title: Identification of multiple integrin ?1 homologs in zebrafish (Danio rerio)
    Article Snippet: Paragraph title: Sequencing of zebrafish integrin β1 paralogs ... PCR reactions were performed using Phusion (New England Biolabs).

    Article Title: Molecular Basis of the Waxy Endosperm Starch Phenotype in Broomcorn Millet (Panicum miliaceum L.)
    Article Snippet: Following further extensive experimentation with primers designed against the S. italica sequence, we used the following protocols to amplify the region corresponding to the entire mature peptide for the S and L loci in the eight plants previously analyzed for starch protein and enzyme activity. .. The L locus was amplified in a single polymerase chain reaction (PCR) using the primers FPSLVVC3 and Rstop3 , in 50 μl volumes using 1× Finnzymes HF buffer (New England Biolabs, Hitchin, Hertfordshire, United Kingdom), 200 μM deoxynucleoside triphosphates (dNTPs), 0.3 μM of each primer, 3% dimethyl sulfoxide, 1 U Finnzymes Phusion High-Fidelity DNA Polymerase (New England Biolabs).

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO. .. Vectors were constructed by USER cloning using plasmid backbones previously presented by [ ]; for details concerning vector construction, see , , and .

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. After ligation the construct pET-21a –AKR and empty vector pET-21a were introduced into E . coli strain BL21 (DE3) for expression studies.

    Article Title: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response
    Article Snippet: The gene for HB36.6 with 40bp of additional pETFLAG overlap sequence, to allow homologous recombination, was assembled via recursive PCR. .. A 10μL volume of outer primer mix was added to 12.7μL of inner primer mix, along with 1μL of 10mM DNTPs, 6μL of 5x Phusion buffer, and 0.3μL of Phusion polymerase (NEB, Waltham, MA) for a final volume of 30μL.

    Selection:

    Article Title: Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24
    Article Snippet: The indexed DNA fragments were enriched for by PCR using AccuPrime (Life Technologies; Grand Island, NY) and Phusion (New England Biolabs, Ipswich, MA) DNA polymerases. .. DNA purification was done using QIAquick and MinElute PCR purification kits (Qiagen; Valencia, CA).

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Paragraph title: In Vitro Selection ... Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Staining:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    cDNA Library Assay:

    Article Title: Identification of multiple integrin ?1 homologs in zebrafish (Danio rerio)
    Article Snippet: PCR reactions were performed using Phusion (New England Biolabs). .. The PCR reactions generated products of ~2.4 kB for β1–1, β1–2 and β1–3, and ~1.9 kB for β1tr-1, β1tr-2 and β1tr-3.

    Activated Clotting Time Assay:

    Article Title: Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation
    Article Snippet: The following master mix was made at room temperature: 5 ul of 5# Phusion HF buffer (New England BioLabs, Ipswich, MA), 0.5 ul of 10 mM dNTPs, 0.75 ul of 10 uM 251envF1 (5%-CAG TCT TTT ATG GTG TAC CAG CTT GGA GGA ATG-3%), 0.75 ul of 10 uM 251envR1 (5%-GAG GAT CCA TCT TCC ACC TCT CCT AAG AGT C-3%), 0.2 ul of Phusion Hot Start high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and 14.8 ul of double-distilled water (ddH2O). .. The following cycling conditions produced the first-round 2,519-bp PCR product: 98°C for 45 s, 35 cycles of 98°C for 15 s and 72°C for 1.5 min, with a 4°C dwell.

    Plasmid Preparation:

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: The overhangs of the megaprimer bind 5′ and 3′ of the insertion site on the target plasmid. .. The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB).

    Article Title: A Ralstonia solanacearum Type III Effector Directs the Production of the Plant Signal Metabolite Trehalose-6-Phosphate
    Article Snippet: Amplifications were performed in two steps, using the cloning primers (731GW-ATG and 731GW-STOP or 731GW-NOSTOP) in the first PCR and then universal primers (attB1 and attB2) in the second PCR, with Platinum Pfx (Life technologies), Pfu Ultra (StrataGen, Kirkland, WA), or Phusion (NEB, Ipswich, MA). .. Expression plasmids (pMP72, pMP93, pMP94, and pMP95) were created by LR Gateway cloning using pAM-PAT-P35S-GW-3xHA (L. D. Noël and J. E. Parker, unpublished data) and used to transform Agrobacterium tumefaciens GV3101::pMP90RK.

    Article Title: A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
    Article Snippet: Paragraph title: PCR, vector construction and protospacers ... Standard reaction volumes were 50 μl including 1x Phusion HF Buffer (New England Biolabs, USA), 0.2 mm dNTPs, 0.4 μM primers (Integrated DNA Technologies (IDT), Belgium), 1 U PfuX7, < 10 ng of gDNA, 3% DMSO.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    SYBR Green Assay:

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: The reaction mixture was neutralized by adding 20 μL of 1 N HCl. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix. .. The amplification primers used were 5′ -AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA-3′ and 5′ -CAAGCAGAAGACGGCATACGA-3′ .

    Multiplex Assay:

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: Paragraph title: Multiplex PCR FFPE DNA Quality Assessment ... PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA).

    Positron Emission Tomography:

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA).

    Agarose Gel Electrophoresis:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB). .. The 2nd RF PCR reaction starts with an initial denaturation step (30 s, 98°C), followed by 35 cycles of denaturation (10 s, 98°C), annealing (30s, Tm (F1/R1) +3°C) and extension (30 s/1 kb, 72°C) with no final extension.

    Article Title: Use of Sequenom Sample ID Plus(R) SNP Genotyping in Identification of FFPE Tumor Samples
    Article Snippet: PCR was carried out using 1 U Phusion HF Taq DNA polymerase and buffer (NEB, Ipswitch, MA), 500 mM KCl, and 10 mM dNTPs (Invitrogen, Carlsbad, CA). .. All PCR reactions were purified using the MinElute PCR Cleanup Kit (QIAGEN, Venlo, Netherlands), and quantified with a Qubit 1.0 fluorimeter (Invitrogen).

    In Vitro:

    Article Title: High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
    Article Snippet: Paragraph title: In Vitro Selection ... Adapter-ligated DNA was purified with the QIAquick PCR Purification Kit and PCR-amplified for 10-13 cycles with Phusion Hot Start Flex DNA Polymerase (NEB) in Buffer HF (NEB) and primers CLTA(#) sel PCR/PE2 short (post-selection) or CLTA(#) lib seq PCR/lib fwd PCR (pre-selection).

    Electrophoresis:

    Article Title: Exponential Megapriming PCR (EMP) Cloning--Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints
    Article Snippet: The 2nd EMP PCR contains 1× HF buffer (NEB), 200 µM of each dNTP, 100 ng–400 ng megaprimer, 25 ng template DNA, and 0.02 U/µL Phusion DNA Polymerase (NEB). .. The 2nd RF PCR reaction starts with an initial denaturation step (30 s, 98°C), followed by 35 cycles of denaturation (10 s, 98°C), annealing (30s, Tm (F1/R1) +3°C) and extension (30 s/1 kb, 72°C) with no final extension.

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix. .. Amplification was monitored by a LightCycler (Bio-Rad) and stopped at the beginning of the saturation point.

    Ethanol Precipitation:

    Article Title: Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features
    Article Snippet: After the 5′ -polyphosphatase treatment, RNA was extracted using phenol-chloroform and ethanol precipitation. .. The cDNA samples were amplified using a mixture of 1 μL of the cDNA, 10 μL of Phusion HF buffer (New England BioLabs, Ipswich, MA), 1 μL of dNTPs (10 mM), 1 μL SYBR green (Qiagen, Valencia, CA), 0.5 μL of HotStart Phusion DNA polymerase (New England BioLabs, Ipswich, MA), and 5 pmole of small RNA PCR primer mix.

    Next-Generation Sequencing:

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Paragraph title: NGS Analysis ... Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs).

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: Paragraph title: Next-generation sequencing (NGS). ... To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl.

    Spectrophotometry:

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: The total genomic DNA was isolated from mosquito midgut tissues using GenElute™ bacterial genomic DNA kit (Sigma, USA) according to manufacturer’s instructions and quantified using a NanoDrop™ 2000 spectrophotometer. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration.

    Produced:

    Article Title: Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation
    Article Snippet: The following master mix was made at room temperature: 5 ul of 5# Phusion HF buffer (New England BioLabs, Ipswich, MA), 0.5 ul of 10 mM dNTPs, 0.75 ul of 10 uM 251envF1 (5%-CAG TCT TTT ATG GTG TAC CAG CTT GGA GGA ATG-3%), 0.75 ul of 10 uM 251envR1 (5%-GAG GAT CCA TCT TCC ACC TCT CCT AAG AGT C-3%), 0.2 ul of Phusion Hot Start high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA), and 14.8 ul of double-distilled water (ddH2O). .. A 23 ul volume of master mix and 2 ul of cDNA or DNA from 5-fold serial dilutions in TE were added to 0.2-ml tubes.

    Concentration Assay:

    Article Title: Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo
    Article Snippet: IGEPal CA-630 was added to a final concentration of 0.1%. .. Libraries were then amplified using Phusion (NEB cat no. F531S) and Illumina Nextera index kit (cat no. FC-121-1011).

    Article Title: Influence of midgut microbiota in Anopheles stephensi on Plasmodium berghei infections
    Article Snippet: Further, the third hypervariable (V3) region of 16S ribosomal RNA gene (approx 280 bp) was amplified (PCR1) from 10 ng genomic DNA using specific primers 341F 5′CCTACGGGAGGCAGCAG3′ and 518R 5′ATTACCGCGGCTGCTGG3′ in a PCR thermocycler. .. The master mix contained 5 µl of 5× Phusion® HF reaction buffer (NEB), 0.4 µl of 10 mM dNTP solution mix (NEB), 0.2 µl of Phusion® HF DNA polymerase (NEB), 2 µl of forward and reverse primers at 10 pM concentration. .. The total reaction volume was made up to 25 µl with nuclease-free water.

    Article Title: TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells
    Article Snippet: Tagmentation was performed in a final volume of 5 μl using 5 ng of purified PCR product, 0.15 μl of Nextera tagment enzyme and tagmentation buffer previously described by Wang et al . .. Tagmented amplicons were then PCR amplified in a final volume of 50 μl using a final concentration of 0.2 mM dNTP (Life Technologies), 0.2 μM Illumina index PCR primers (Integrated DNA Technologies), 1x Phusion DNA polymerase buffer (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs). .. PCR cycling conditions used were as follows: 72 °C for 3 minutes, 98 °C for 2 minutes and 15 cycles of 98 °C for 10 seconds, 63 °C for 30 secconds, and 72 °C for 3 minutes.

    Article Title: Free-Chlorine Disinfection as a Selection Pressure on Norovirus
    Article Snippet: Ligation products were purified by the Agencourt AMPureXP system (Beckman Coulter, Brea, CA), and DNA concentration was measured by the Quantus fluorometer using the QuantiFluor dsDNA System (Promega). .. To enrich the DNA libraries, a second PCR was performed in a 10-μl reaction mixture with Protocol Phusion high-fidelity PCR master mix with 5 μl HF buffer (New England BioLabs), 0.5 μl Kapa Illumina primer P1 (5′-AAT GAT ACG GCG ACC GA-3′) (10 μM), 0.5 μl Illumina primer P2 (5′-CAA GCA GAA GAC GGC ATA CGA-3′) (10 μM), 3 μl each ligated DNA, and PCR-grade water up to 10 μl.

    Article Title: Rapid targeted gene disruption in Bacillus anthracis
    Article Snippet: The resulting insertion sites are shown in Table and the mutations necessary to retarget the intron to a desired site were introduced through PCR mutagenesis using two pairs of partially overlapping primers (Table ) to assemble a short DNA fragment flanked by HindIII and BsrG1 sites and incorporating the changes within the intron required to redirect the intron to a new locus as described previously [ ]. .. Briefly, for each mutant intron to be created 10 cycles of PCR was performed using 2 μL of the mutant specific IBS and EBS universal primer at a concentration of 100 μM and 10 μM respectively in a 20 uL reaction using Phusion® (NEB) PCR mastermix and 0.2 ng of wild-type intron template. .. In a separate tube a similar reaction was performed using mutant specific primers EBS1delta (100 μM) and EBS2 (10 μM) primers.

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: PCR was done with a temperature program starting at 98°C for 30 sec, followed by 30 cycles of 98°C for 10 sec, 52°C for 30 sec, 72°C for 30 sec and a final elongation at 72°C for 7 min. .. The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. The amplified product was gel purified using a QIAquick gel extraction kit (Qiagen).

    Lysis:

    Article Title: Cloning of upstream region and cellulose synthase operon genes involved in bacterial cellulose biosynthesis by Gluconacetobacter hansenii ATCC23769
    Article Snippet: Extraction of bacterial genomic DNA was carried out using cell lysis under heating in the presence of sodium dodecyl sulfate (SDS) and proteinase K, followed by purification with a phenol:chloroform:isoamylic alcohol solution (PCI) and subsequent dialysis in TE buffer pH 8.0. .. The cloning of the genes related to the production of bacterial cellulose was carried out with amplification reactions (PCR) using specifically designed primers in the presence of Pfx Plantinum (Invitrogen) or Phusion (New England Biolabs) polymerases.

    Recombinant:

    Article Title: A Novel Aldo-Keto Reductase (AKR17A1) of Anabaena sp. PCC 7120 Degrades the Rice Field Herbicide Butachlor and Confers Tolerance to Abiotic Stresses in E. coli
    Article Snippet: The reaction was performed with a final concentration of 100ng DNA, 5μl of 5× Phusion HF Buffer (NEB), 200μM dNTPs, 0.5μM forward and reverse primer and 0.5U Phusion DNA polymerase (NEB) in an iCycler (Bio-Rad, USA). .. After ligation the construct pET-21a –AKR and empty vector pET-21a were introduced into E . coli strain BL21 (DE3) for expression studies.

    Homologous Recombination:

    Article Title: A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response
    Article Snippet: The gene for HB36.6 with 40bp of additional pETFLAG overlap sequence, to allow homologous recombination, was assembled via recursive PCR. .. A 10μL volume of outer primer mix was added to 12.7μL of inner primer mix, along with 1μL of 10mM DNTPs, 6μL of 5x Phusion buffer, and 0.3μL of Phusion polymerase (NEB, Waltham, MA) for a final volume of 30μL.

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  • 99
    New England Biolabs phusion high fidelity dna polymerase phusion
    Proofreading <t>DNA</t> polymerases can be inhibited by certain primers. ( A ) Example of inhibitory primer using PSGXL. A slightly longer target DNA fragment can be successfully amplified by the primers OuterR and Stat3F, but use Stat3R instead of OuterR caused PCR failure. ( B ) Primers Stat3R and GfapR are inhibitory to PSGXL and PS, but not to LATaq and Taq. The 2 kb targets were amplified using primers Olig2F and Olig2R, additional primer Olig2.6F, Stat3R, GfapR were added to the reaction. ( C ) Primers Stat3R and GfapR are inhibitory to all the tested proofreading DNA polymerases. Primers pBSIIF and pBSIIR were used to amplify the entire plasmid pBlueScript II KS (−). Adding the additional primer Stat3R or GfapR substantially reduced the PCR yield using the proofreading DNA polymerases such as <t>Phusion,</t> Q5, Cobuddy, PS, PSGXL and PfuFly. Arrowheads indicate target DNA fragments.
    Phusion High Fidelity Dna Polymerase Phusion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase phusion/product/New England Biolabs
    Average 99 stars, based on 826 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity dna polymerase phusion - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs phusion high fidelity master mix
    Proofreading <t>DNA</t> polymerases can be inhibited by certain primers. ( A ) Example of inhibitory primer using PSGXL. A slightly longer target DNA fragment can be successfully amplified by the primers OuterR and Stat3F, but use Stat3R instead of OuterR caused PCR failure. ( B ) Primers Stat3R and GfapR are inhibitory to PSGXL and PS, but not to LATaq and Taq. The 2 kb targets were amplified using primers Olig2F and Olig2R, additional primer Olig2.6F, Stat3R, GfapR were added to the reaction. ( C ) Primers Stat3R and GfapR are inhibitory to all the tested proofreading DNA polymerases. Primers pBSIIF and pBSIIR were used to amplify the entire plasmid pBlueScript II KS (−). Adding the additional primer Stat3R or GfapR substantially reduced the PCR yield using the proofreading DNA polymerases such as <t>Phusion,</t> Q5, Cobuddy, PS, PSGXL and PfuFly. Arrowheads indicate target DNA fragments.
    Phusion High Fidelity Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity master mix/product/New England Biolabs
    Average 99 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity master mix - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    79
    New England Biolabs rapid cycle compatible phusion high fidelity polymerase
    E-Gel image showing sequential 26-cycle <t>Phusion</t> / GAPDH cleaning and decontamination experiments. Odd numbered lanes are identical positive controls preceded by cleaning protocol A. Lanes (M) 50 bp ladder, (2) NTC preceded by cleaning protocol A, (4) NTC preceded by cleaning protocol B, (6) NTC preceded by cleaning protocol C, (8) NTC preceded by cleaning protocol D, (10) NTC preceded by cleaning protocol E. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes.
    Rapid Cycle Compatible Phusion High Fidelity Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rapid cycle compatible phusion high fidelity polymerase/product/New England Biolabs
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rapid cycle compatible phusion high fidelity polymerase - by Bioz Stars, 2019-10
    79/100 stars
      Buy from Supplier

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    Proofreading DNA polymerases can be inhibited by certain primers. ( A ) Example of inhibitory primer using PSGXL. A slightly longer target DNA fragment can be successfully amplified by the primers OuterR and Stat3F, but use Stat3R instead of OuterR caused PCR failure. ( B ) Primers Stat3R and GfapR are inhibitory to PSGXL and PS, but not to LATaq and Taq. The 2 kb targets were amplified using primers Olig2F and Olig2R, additional primer Olig2.6F, Stat3R, GfapR were added to the reaction. ( C ) Primers Stat3R and GfapR are inhibitory to all the tested proofreading DNA polymerases. Primers pBSIIF and pBSIIR were used to amplify the entire plasmid pBlueScript II KS (−). Adding the additional primer Stat3R or GfapR substantially reduced the PCR yield using the proofreading DNA polymerases such as Phusion, Q5, Cobuddy, PS, PSGXL and PfuFly. Arrowheads indicate target DNA fragments.

    Journal: Scientific Reports

    Article Title: Guanine-rich sequences inhibit proofreading DNA polymerases

    doi: 10.1038/srep28769

    Figure Lengend Snippet: Proofreading DNA polymerases can be inhibited by certain primers. ( A ) Example of inhibitory primer using PSGXL. A slightly longer target DNA fragment can be successfully amplified by the primers OuterR and Stat3F, but use Stat3R instead of OuterR caused PCR failure. ( B ) Primers Stat3R and GfapR are inhibitory to PSGXL and PS, but not to LATaq and Taq. The 2 kb targets were amplified using primers Olig2F and Olig2R, additional primer Olig2.6F, Stat3R, GfapR were added to the reaction. ( C ) Primers Stat3R and GfapR are inhibitory to all the tested proofreading DNA polymerases. Primers pBSIIF and pBSIIR were used to amplify the entire plasmid pBlueScript II KS (−). Adding the additional primer Stat3R or GfapR substantially reduced the PCR yield using the proofreading DNA polymerases such as Phusion, Q5, Cobuddy, PS, PSGXL and PfuFly. Arrowheads indicate target DNA fragments.

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

    Techniques: Amplification, Polymerase Chain Reaction, Plasmid Preparation

    Engineered proofreading DNA polymerases have good performance. ( A ) Testing various DNA polymerases for the amplification of a 1 kb fragment with 70% GC-content. ( B ) Testing different DNA polymerases for the amplification of an 18 kb fragment from λ DNA. ( C ) Amplification of the ORF of insulin-like growth factor I receptor (Igf1r) from mouse cDNA. The 3′ untranslated region of Igf1r is longer than 7 kb. ( D ) Amplification of the entire LCas9 plasmid. The 10.9 kb LCas9 plasmid contains a high GC-rich region. Pfu: Pfu DNA polymerase. LATaq: LA Taq Version 2.0. TransHF: TransTaq DNA polymerase High Fidelity. Phusion: Phusion High-Fidelity DNA polymerase. Q5: Q5 High-Fidelity DNA polymerase. Cobuddy: Cobuddy Super Fidelity DNA polymerase. PSGXL: PrimeSTAR GXL DNA polymerase. PfuFly: TransStart FastPfu Fly DNA polymerase. Enh: 0–10 μl of GC enhancer was added into the 25 μl PCR reagent.

    Journal: Scientific Reports

    Article Title: Guanine-rich sequences inhibit proofreading DNA polymerases

    doi: 10.1038/srep28769

    Figure Lengend Snippet: Engineered proofreading DNA polymerases have good performance. ( A ) Testing various DNA polymerases for the amplification of a 1 kb fragment with 70% GC-content. ( B ) Testing different DNA polymerases for the amplification of an 18 kb fragment from λ DNA. ( C ) Amplification of the ORF of insulin-like growth factor I receptor (Igf1r) from mouse cDNA. The 3′ untranslated region of Igf1r is longer than 7 kb. ( D ) Amplification of the entire LCas9 plasmid. The 10.9 kb LCas9 plasmid contains a high GC-rich region. Pfu: Pfu DNA polymerase. LATaq: LA Taq Version 2.0. TransHF: TransTaq DNA polymerase High Fidelity. Phusion: Phusion High-Fidelity DNA polymerase. Q5: Q5 High-Fidelity DNA polymerase. Cobuddy: Cobuddy Super Fidelity DNA polymerase. PSGXL: PrimeSTAR GXL DNA polymerase. PfuFly: TransStart FastPfu Fly DNA polymerase. Enh: 0–10 μl of GC enhancer was added into the 25 μl PCR reagent.

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

    Techniques: Amplification, Gas Chromatography, Plasmid Preparation, Polymerase Chain Reaction

    Proofreading DNA polymerases but not Taq DNA polymerase bind to G-rich sequences. Biotin-labeled oligonucleotide Bio-G6 was subjected to electrophoresis mobility shift assay. Electrophoresis of the Bio-G6 alone showed 3 bands in the gel, as the 6 consecutive G in Bio-G6 tends to form intermolecular G-quadruplex. Taq didn’t cause any band shift. However, when the proofreading DNA polymerase Phusion or PSGXL was added, the bands corresponding to intermolecular G-quadruplex were retarded. The band shift caused by proofreading DNA polymerases were disappeared by adding excess amount of competitor G6 but not non-specific competitor MG. Note that the anti-PSGXL antibody also caused a weak supershift.

    Journal: Scientific Reports

    Article Title: Guanine-rich sequences inhibit proofreading DNA polymerases

    doi: 10.1038/srep28769

    Figure Lengend Snippet: Proofreading DNA polymerases but not Taq DNA polymerase bind to G-rich sequences. Biotin-labeled oligonucleotide Bio-G6 was subjected to electrophoresis mobility shift assay. Electrophoresis of the Bio-G6 alone showed 3 bands in the gel, as the 6 consecutive G in Bio-G6 tends to form intermolecular G-quadruplex. Taq didn’t cause any band shift. However, when the proofreading DNA polymerase Phusion or PSGXL was added, the bands corresponding to intermolecular G-quadruplex were retarded. The band shift caused by proofreading DNA polymerases were disappeared by adding excess amount of competitor G6 but not non-specific competitor MG. Note that the anti-PSGXL antibody also caused a weak supershift.

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

    Techniques: Labeling, Electrophoresis, Mobility Shift, Electrophoretic Mobility Shift Assay

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0057792

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

    Article Snippet: PCR conditions for the 50 µl reactions were as follows: 1X Phusion High Fidelity (GC) buffer (New England Biolabs), 3% DMSO, 0.1 mM dNTPs, 0.02 units/µl Phusion DNA polymerase (New England Biolabs), 0.4 pmol/µl of each and 1.5 ng/µl plasmid DNA template.

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

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0057792

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

    Article Snippet: PCR conditions for the 50 µl reactions were as follows: 1X Phusion High Fidelity (GC) buffer (New England Biolabs), 3% DMSO, 0.1 mM dNTPs, 0.02 units/µl Phusion DNA polymerase (New England Biolabs), 0.4 pmol/µl of each and 1.5 ng/µl plasmid DNA template.

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

    E-Gel image showing sequential 26-cycle Phusion / GAPDH cleaning and decontamination experiments. Odd numbered lanes are identical positive controls preceded by cleaning protocol A. Lanes (M) 50 bp ladder, (2) NTC preceded by cleaning protocol A, (4) NTC preceded by cleaning protocol B, (6) NTC preceded by cleaning protocol C, (8) NTC preceded by cleaning protocol D, (10) NTC preceded by cleaning protocol E. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes.

    Journal: PLoS ONE

    Article Title: The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

    doi: 10.1371/journal.pone.0118182

    Figure Lengend Snippet: E-Gel image showing sequential 26-cycle Phusion / GAPDH cleaning and decontamination experiments. Odd numbered lanes are identical positive controls preceded by cleaning protocol A. Lanes (M) 50 bp ladder, (2) NTC preceded by cleaning protocol A, (4) NTC preceded by cleaning protocol B, (6) NTC preceded by cleaning protocol C, (8) NTC preceded by cleaning protocol D, (10) NTC preceded by cleaning protocol E. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes.

    Article Snippet: Rapid Single-Plex Amplification: A protocol based on the rapid cycle-compatible Phusion high fidelity polymerase (New England Biolabs, Ipswitch, MA) was employed to evaluate the operation of the rzPCR system for single-plex PCR.

    Techniques: Positive Control

    RZTC power and temperature history from startup for six Phusion rzPCR experiments with thermal cycling intervals indicated by the shaded bands. (A) Total (wall) power drawn by the heater control box during initial idle, warm-up, and cycling. (B) Detail of power fluctuations during cleaning, sample load, amplification, and sample unload. (C) Temperature fluctuations for heater blocks 2–4 about their set-points at 60°C, 72°C, and 98°C. (D) Temperature evolution of unheated block 1 over time.

    Journal: PLoS ONE

    Article Title: The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

    doi: 10.1371/journal.pone.0118182

    Figure Lengend Snippet: RZTC power and temperature history from startup for six Phusion rzPCR experiments with thermal cycling intervals indicated by the shaded bands. (A) Total (wall) power drawn by the heater control box during initial idle, warm-up, and cycling. (B) Detail of power fluctuations during cleaning, sample load, amplification, and sample unload. (C) Temperature fluctuations for heater blocks 2–4 about their set-points at 60°C, 72°C, and 98°C. (D) Temperature evolution of unheated block 1 over time.

    Article Snippet: Rapid Single-Plex Amplification: A protocol based on the rapid cycle-compatible Phusion high fidelity polymerase (New England Biolabs, Ipswitch, MA) was employed to evaluate the operation of the rzPCR system for single-plex PCR.

    Techniques: Amplification, Blocking Assay

    E-Gel image showing alternating positive control and no template control 26-cycle GAPDH / Phusion experiments. Lanes (M) 50 bp ladder, (1) Bench-top NTC, (2) Bench-top positive control, (3, 5, 7, 9) rzPCR NTC, (4, 6, 8, 10) rzPCR positive control. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes. Streaks and smudging are post-separation handling artifacts.

    Journal: PLoS ONE

    Article Title: The Rotary Zone Thermal Cycler: A Low-Power System Enabling Automated Rapid PCR

    doi: 10.1371/journal.pone.0118182

    Figure Lengend Snippet: E-Gel image showing alternating positive control and no template control 26-cycle GAPDH / Phusion experiments. Lanes (M) 50 bp ladder, (1) Bench-top NTC, (2) Bench-top positive control, (3, 5, 7, 9) rzPCR NTC, (4, 6, 8, 10) rzPCR positive control. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes. Streaks and smudging are post-separation handling artifacts.

    Article Snippet: Rapid Single-Plex Amplification: A protocol based on the rapid cycle-compatible Phusion high fidelity polymerase (New England Biolabs, Ipswitch, MA) was employed to evaluate the operation of the rzPCR system for single-plex PCR.

    Techniques: Positive Control