phusion  (New England Biolabs)


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    Name:
    Phusion GC Buffer Pack
    Description:
    Phusion GC Buffer Pack 6 0 ml
    Catalog Number:
    b0519s
    Price:
    24
    Size:
    6 0 ml
    Category:
    Buffers
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    New England Biolabs phusion
    Phusion GC Buffer Pack
    Phusion GC Buffer Pack 6 0 ml
    https://www.bioz.com/result/phusion/product/New England Biolabs
    Average 99 stars, based on 367 article reviews
    Price from $9.99 to $1999.99
    phusion - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) 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

    2) 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

    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

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Analysis of intestinal microbial communities of cerebral infarction and ischemia patients based on high throughput sequencing technology and glucose and lipid metabolism
    Article Snippet: .. The sequences were forward, 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse, 5′-GGACTACHVGGGTWTCTAAT-3′, and the reaction was performed using Phusion High-Fidelity PCR master mix with GC buffer (New England Biolabs, Inc., Ipswich, MA, USA). .. Samples were sequenced by Illumina MiSeq platform provided by Illumina, Inc. (San Diego, CA, USA) with the following conditions: Denaturation 1 min at 98°C, 10 sec at 98°C, 30 sec at 50°C and 30 sec at 72°Cfor 30 cycles, and 72°C at 5 min.

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
    Article Snippet: .. The forward and reverse primers (5 μl each of a 10 μM solution), the pGFPuv template (1 μl of a 0.1 ng/μl solution), dNTPs (2 μl each of a 1 mM solution), Phusion GC Buffer (5 μl of a 10× stock, New England Biolabs), Phusion DNA Polymerase (1 μl, two units) and dH2 O (31 μl) were mixed and subjected to the following PCR program: 95°C (2 min), followed by 40 cycles of 95°C (30 s), 40°C (60 s) and 72°C (3.3 min), with a final extension at 72°C (2 min). .. An identical PCR reaction was then repeated, using 5 μl of the previous reaction as the template, followed by purification with a PCR cleanup kit (Promega).

    Article Title: SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing
    Article Snippet: .. A 50 μl PCR reaction contained 2.5 μl of cDNA template, 1 μl of 100 μM forward and reverse primers, 1 μl of 10 mM dNTPs, 10 μl 5× Phusion Buffer, 33.5 μl water and 1 U Phusion DNA polymerase (NEB). .. Multiple reactions were made together and split before the pertinent number of PCR amplification cycles.

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *
    Article Snippet: .. End Point and Quantitative-PCR (qPCR) Analysis of β-GT-treated MspI- or HpaII-digested DNA End point PCR was completed with Phusion-GC (NEB) polymerase master mix. ..

    Article Title: Identification of Fungal Communities Associated with the Biodeterioration of Waterlogged Archeological Wood in a Han Dynasty Tomb in China
    Article Snippet: .. All PCR reactions were carried out using the Phusion® High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, United Kingdom). .. Amplifications were carried out in a 30 μL mixture that included 15 μL of Master Mix (2X), a 0.5 μM final concentration of the forward and reverse primers, 10 ng of template DNA and nuclease-free water to 30 μL.

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach
    Article Snippet: .. 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.

    Article Title: Land use impacts poison frog chemical defenses through changes in leaf litter ant communities
    Article Snippet: .. For all reactions, we used 2 μL of each primer (10 μM) and 25 μL of 2X Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Ipswich, MA, USA) in a total reaction volume of 50 μL. .. We used a touchdown PCR program to amplify CO1 as follows: 95°C for 5 min; 5 cycles of 95°C for 30 s, 45°C for 30 s with −1°C per cycle, 72°C for 1 min; and 40 rounds of 95°C for 30 s, 40°C for 30 s, and 72°C for 1 min; ending with a single incubation of 72°C for 5 min. PCR reactions were stored at −20°C for one week and then run on a 1% SYBRSafe/agarose gel (Life Technologies).

    Real-time Polymerase Chain Reaction:

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *
    Article Snippet: .. End Point and Quantitative-PCR (qPCR) Analysis of β-GT-treated MspI- or HpaII-digested DNA End point PCR was completed with Phusion-GC (NEB) polymerase master mix. ..

    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.

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    New England Biolabs neb phusion polymerase
    Validation and testing of PrimerMapper. ( a ) Primers were designed using PrimerMapper to span the C. elegans transcript, ZK5204a, and PCR products from a series of PCR reactions using these primers were examined by gel electrophoresis. Primer sequences are displayed in Table 1 . Robust bands of the correct size were obtained from each PCR. The first and last lanes are a GenRuler DNA ladder mix (Thermo Fisher Scientific, Waltham, MA). Primer parings starting from the left second lane were: F2 + R1, F2 + R2, F2 + R3, F2 + R4, F2 + R5, F2 + R6, F3 + R1, F3 + R2, F3 + R3, F3 + R4, F1 + R2, F1 + R3, F1 + R4, F1 + R5, F1 + R6, F1 + R8, F3 + R9, F5 + R6. ( b ) Files containing different numbers of sequences (2, 10, 20, 50, 100, 150, 200, 1,000) were provided as input to PrimerMapper and ran using default settings to generate primer text files for each sequence and graphic file generation input text files (i.e. Step 1 of execution – see Fig. 2 ). The resulting data is plotted with time (seconds) on the x-axis and the number of sequences on the y-axis. Fitting the relationship between sequence number and run-time with a quadratic equation yields an R 2 value of 0.9995. ( c–f) Comparison of the melting temperatures obtained for PrimerMapper from 100 randomly generated primers (18–30bps in size) with that of the <t>NEB</t> calculator for NEB Taq DNA Polymerase (c) , NEB <t>Phusion</t> ® Polymerase ( d ), NEB Q5 ® Hi-Fi Polymerase (e) , and with Primer3 16 ( f ).
    Neb Phusion Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion high fidelity dna polymerase
    The median CEL intensities for each amplicon obtained by using Stoffel <t>DNA</t> polymerase and <t>Phusion</t> DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3841 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion hf pcr reactions
    Schematics of the subcycling <t>PCR</t> protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. <t>Phusion</t> and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
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    Validation and testing of PrimerMapper. ( a ) Primers were designed using PrimerMapper to span the C. elegans transcript, ZK5204a, and PCR products from a series of PCR reactions using these primers were examined by gel electrophoresis. Primer sequences are displayed in Table 1 . Robust bands of the correct size were obtained from each PCR. The first and last lanes are a GenRuler DNA ladder mix (Thermo Fisher Scientific, Waltham, MA). Primer parings starting from the left second lane were: F2 + R1, F2 + R2, F2 + R3, F2 + R4, F2 + R5, F2 + R6, F3 + R1, F3 + R2, F3 + R3, F3 + R4, F1 + R2, F1 + R3, F1 + R4, F1 + R5, F1 + R6, F1 + R8, F3 + R9, F5 + R6. ( b ) Files containing different numbers of sequences (2, 10, 20, 50, 100, 150, 200, 1,000) were provided as input to PrimerMapper and ran using default settings to generate primer text files for each sequence and graphic file generation input text files (i.e. Step 1 of execution – see Fig. 2 ). The resulting data is plotted with time (seconds) on the x-axis and the number of sequences on the y-axis. Fitting the relationship between sequence number and run-time with a quadratic equation yields an R 2 value of 0.9995. ( c–f) Comparison of the melting temperatures obtained for PrimerMapper from 100 randomly generated primers (18–30bps in size) with that of the NEB calculator for NEB Taq DNA Polymerase (c) , NEB Phusion ® Polymerase ( d ), NEB Q5 ® Hi-Fi Polymerase (e) , and with Primer3 16 ( f ).

    Journal: Scientific Reports

    Article Title: PrimerMapper: high throughput primer design and graphical assembly for PCR and SNP detection

    doi: 10.1038/srep20631

    Figure Lengend Snippet: Validation and testing of PrimerMapper. ( a ) Primers were designed using PrimerMapper to span the C. elegans transcript, ZK5204a, and PCR products from a series of PCR reactions using these primers were examined by gel electrophoresis. Primer sequences are displayed in Table 1 . Robust bands of the correct size were obtained from each PCR. The first and last lanes are a GenRuler DNA ladder mix (Thermo Fisher Scientific, Waltham, MA). Primer parings starting from the left second lane were: F2 + R1, F2 + R2, F2 + R3, F2 + R4, F2 + R5, F2 + R6, F3 + R1, F3 + R2, F3 + R3, F3 + R4, F1 + R2, F1 + R3, F1 + R4, F1 + R5, F1 + R6, F1 + R8, F3 + R9, F5 + R6. ( b ) Files containing different numbers of sequences (2, 10, 20, 50, 100, 150, 200, 1,000) were provided as input to PrimerMapper and ran using default settings to generate primer text files for each sequence and graphic file generation input text files (i.e. Step 1 of execution – see Fig. 2 ). The resulting data is plotted with time (seconds) on the x-axis and the number of sequences on the y-axis. Fitting the relationship between sequence number and run-time with a quadratic equation yields an R 2 value of 0.9995. ( c–f) Comparison of the melting temperatures obtained for PrimerMapper from 100 randomly generated primers (18–30bps in size) with that of the NEB calculator for NEB Taq DNA Polymerase (c) , NEB Phusion ® Polymerase ( d ), NEB Q5 ® Hi-Fi Polymerase (e) , and with Primer3 16 ( f ).

    Article Snippet: In each case, there were robust correlations observed between the melting temperatures calculated by PrimerMapper and each algorithm, with the highest correlation observed for PrimerMapper to that of the NEB calculator for NEB Phusion® polymerase ( ; r 2 = 0.99).

    Techniques: Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Generated

    The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

    doi: 10.1073/pnas.0803240105

    Figure Lengend Snippet: The median CEL intensities for each amplicon obtained by using Stoffel DNA polymerase and Phusion DNA polymerase in the gap-fill reaction are plotted against each other. The CEL intensities that were

    Article Snippet: The extension was performed by addition of 0.4 units of Phusion High-Fidelity DNA Polymerase (New England Biolabs), 3 μl 1.0 mM dNTP, 5 units Ampligase (Epicenter Biotechnologies) in a 15-μl volume at 60°C for 15 min followed by 72°C for 15 min.

    Techniques: Amplification

    Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Journal: Nucleic Acid Therapeutics

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    doi: 10.1089/nat.2014.0513

    Figure Lengend Snippet: Polyacrylamide gel electrophoresis (PAGE) electrophoresis of the polymerase–endonuclease amplification reaction (PEAR) products. Lowercase letters (agct) represents unmodified dNTPs; uppercase letters (AGCT) represent modified dNTPs (2′-F-dNTPs or dNTPαSs). (A) PEAR by Phusion DNA polymerase using unmodified dNTPs, 2′-F-modified dATP and dGTP, respectively. Lane 1: 10-bp DNA ladder; lane 2: normal dNTPs; lane 3: 2′-F-dATP modified PEAR products; lane 4: control without PspGI; lane 5: control without Phusion DNA polymerase; lane 6: control without dATP; lane 7: 10-bp DNA ladder; lane 8: 2′-F-dGTP modified PEAR products; lane 9: control without PspGI; lane 10 : control without Phusion DNA polymerase; lane 11: control without dGTP. (B) PEAR by Phusion DNA polymerase using 2′-F-dCTP and 2′-F-dUTP. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without Phusion DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without Phusion DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (C) 2′-F-dATP and 2′-F-dGTP modified PEAR products as “seeds” for PEAR. Lane 1: 10-bp DNA ladder; lane 2: control without PspGI; lane 3: using 2′-F-dATP modified PEAR products as “seeds” for PEAR; lane 4: control without PspGI; lane 5: using 2′-F-dGTP modified PEAR products as seeds for PEAR. (D) 2′-F-dATP and 2′-F-dGTP modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dGTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase ; lane 9: control without 2′-F-dGTP; lane 10: 10-bp DNA ladder. (E) PEAR amplification of 2′-F-dCTP and 2′-F-dUTP modified products using KOD DNA polymerase. Lane 1: 2′-F-dCTP modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dCTP; lane 5: 10-bp DNA ladder; lane 6: 2′-F-dUTP modified PEAR products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without dUTP; lane 10: 10-bp DNA ladder. (F) PEAR amplification of dTTPαS modified and 2′-F-dATP+dGTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: dTTPαS modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without dTTPαS; lane 5: 20 bp DNA ladder; lane 6: 2′-F-dATP and dGTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dATP and dGTPαS; lane 10: 20-bp DNA ladder. (G) PEAR amplification of 2′-F-dATP+dCTPαS double modified and 2′-F-dATP+dTTPαS double modified PEAR products using KOD DNA polymerase. Lane 1: 2′-F-dATP+dCTPαS double modified PEAR products; lane 2: control without PspGI; lane 3: control without KOD DNA polymerase; lane 4: control without 2′-F-dATP and dCTPαS; lane 5: 20-bp DNA ladder; lane 6: 2′-F-dATP+dTTPαS double modified PEAR amplified products; lane 7: control without PspGI; lane 8: control without KOD DNA polymerase; lane 9: control without 2′-F-dGTP and dTTPαS; lane 10: 20-bp DNA ladder.

    Article Snippet: Four 2′-fluoro-2′-deoxyribinucleoside-5′-triphosphates (2′-F-dNTPs), including 2′-F-dATP, 2′-F-dCTP, 2′-F-dGTP, 2′-F-dUTP and four 2′-deoxyribonucleotides-5′-O-(1-thiotriphosphate) (dNTPαSs), including dATPαS, dGTPαS, dCTPαS, and dTTPαS, whose structural formula are shown in , were purchased from Trilink BioTechnologies, Inc. KOD DNA polymerase was purchased from TOYOBO (Shanghai) Biotech Co., Ltd. Phusion DNA polymerase, highly thermostable restriction enzyme PspGI, and dNTPs were purchased from New England Biolabs, Inc. UNIQ-10 Spin Column Oligo DNA Purification Kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Amplification, Modification

    Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.

    Journal: PLoS ONE

    Article Title: Improved PCR Amplification of Broad Spectrum GC DNA Templates

    doi: 10.1371/journal.pone.0156478

    Figure Lengend Snippet: Schematics of the subcycling PCR protocol. A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.

    Article Snippet: To address high GC content various additives were included in the Phusion HF PCR reactions as follows: 7-deaza-dGTP (NEB) at a 40:60 ratio with normal dGTP, as well as 50:50 and 60:40 ratios keeping the final concentration of dNTPs constant; DMSO (Sigma) at a final concentration of 2.5%, 5%, and 10%; betaine (Sigma) at a final concentration of 1M, 2M and 4M.

    Techniques: Polymerase Chain Reaction, Amplification, Electrophoresis, Chromatin Immunoprecipitation, Purification