phusion (New England Biolabs)

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Name:

Phusion HF Buffer Pack

Description:

Phusion HF Buffer Pack 6 0 ml

Catalog Number:

B0518S

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Category:

Buffers

Size:

6 0 ml

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New England Biolabs phusion

Phusion HF Buffer Pack 6 0 ml
https://www.bioz.com/result/phusion/product/New England Biolabs
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99

phusion - by Bioz Stars, 2021-07

86/100 stars

Images

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-018-24677-5

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

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

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

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

Article Title: Dissecting and Tuning Primer Editing by Proofreading Polymerases

Journal: bioRxiv

doi: 10.1101/2021.05.11.443694

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

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

Techniques Used: Amplification

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv036

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

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

Techniques Used: Activity Assay, Selection, Construct

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

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

Journal: Scientific Reports

doi: 10.1038/s41598-019-41235-9

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

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

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

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks396

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

Polymerase Chain Reaction:

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

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

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

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

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

Hybridization:

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

Clone Assay:

DNA Sequencing:

Concentration Assay:

Polyacrylamide Gel Electrophoresis:

Purification:

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