high fidelity dna polymerase  (New England Biolabs)


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    Name:
    Phusion High Fidelity DNA Polymerase
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    Phusion High Fidelity DNA Polymerase 500 units
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    m0530l
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    500 units
    Category:
    Thermostable DNA Polymerases
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    New England Biolabs high fidelity dna polymerase
    Phusion High Fidelity DNA Polymerase
    Phusion High Fidelity DNA Polymerase 500 units
    https://www.bioz.com/result/high fidelity dna polymerase/product/New England Biolabs
    Average 99 stars, based on 120 article reviews
    Price from $9.99 to $1999.99
    high fidelity dna polymerase - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease"

    Article Title: Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.16-20296

    Cas9-mediated indel formation and DNA sequencing. ( A ) T7E1 mismatch detection assays demonstrating frequency of indel formation (%indel) for each gRNA targeting VEGF-A, expressed in mean ± standard deviation. Lentiviral vectors expressing SpCas9 without VEGF-targeted gRNAs were used as controls. Representative results are shown from four total independent experiments. ( B ) Sanger sequencing chromatograms showing indel formation at the predicted cut site for each target site. ( C ) Representative deep sequencing results for V-1 confirming indel formation at the predicted cut site ( red arrowhead ), including insertions ( bases in red ) and deletions ( dashes ).
    Figure Legend Snippet: Cas9-mediated indel formation and DNA sequencing. ( A ) T7E1 mismatch detection assays demonstrating frequency of indel formation (%indel) for each gRNA targeting VEGF-A, expressed in mean ± standard deviation. Lentiviral vectors expressing SpCas9 without VEGF-targeted gRNAs were used as controls. Representative results are shown from four total independent experiments. ( B ) Sanger sequencing chromatograms showing indel formation at the predicted cut site for each target site. ( C ) Representative deep sequencing results for V-1 confirming indel formation at the predicted cut site ( red arrowhead ), including insertions ( bases in red ) and deletions ( dashes ).

    Techniques Used: DNA Sequencing, Standard Deviation, Expressing, Sequencing

    2) Product Images from "Targeted recombination between homologous chromosomes for precise breeding in tomato"

    Article Title: Targeted recombination between homologous chromosomes for precise breeding in tomato

    Journal: Nature Communications

    doi: 10.1038/ncomms15605

    Tomato fruit colour DSB repair assay. ( a ) Crossing yellow flesh e 3756 35S:Cas9 and bicolor cc383 u6-26:Ps#1-sgRNA gives F 1 plants with a pale Bicolour fruit phenotype. F 1 plants expressing both Cas9 and gRNA were selected. The gRNA was designed for DSB induction (black lightning) in both alleles between the yellow flesh e 3756 and bicolor cc383 mutations (*). In case of error-prone NHEJ repair (blue line) of bicolor cc383 , fruit colour is yellow. In cases of non-crossover or crossover, fruit colour is expected to be red or bicolour or yellow with red spots in case of late event. Note that whole red fruits were not obtained. Rather, fruits with red spots in a yellow or bicolour background were found and are shown together with additional products of HR-induced repair in Supplementary Fig. 1 . ( b ) Fruit phenotype distribution in F 1 plants and control: Bicolour fruits are shown as orange boxes; Yellow fruits as yellow; Fruits with red sectors (putative somatic HR) are shown as red-stripped boxes. Each column represents a fruit population derived from cross of independent transgenic lines of Cas9 and a given u6-26:Ps#1-sgRNA line. The number of fruits analysed is shown on the column in black. ( c ) Sequences of the NHEJ DSB repair footprints and their relative frequency are shown. The CRISPR-Cas target sequence from the PSY1 is shown on the top. The PSY1 start codon is shown in red and the PAM in blue. The top pie represents an average of illumina Hiseq reads from 22 different F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA. The low pie represents an average of ilummina Hiseq reads from two plants of control F 1 population ( yellow flesh e 3756 × bicolor cc383 F 1 plants with no CRISPR-Cas). ( d ) Inverse PCR scheme for identification of recombinant DNA fragments (details in Supplementary Fig. 4 ). (1) DNA from separate leaves was digested with ApaI(A) and HindIII(H) and then blunted. (2) Each sample was self-ligated, and (3) amplified by two different primer sets (green and yellow). Blue- Bicolor ; red- Yellow flesh ; Dashed blue line- Bicolor deletion, *- Yellow flesh mutation. ( e ) Ratio of parental (P) versus recombinant (R) types (obtained from panel C) in individual plants. Plants 1–15- F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA; Plant 16- synthetic crossover control; Plants17–18-Yellow flesh × Bicolor (Cas9-) F 1 plants.
    Figure Legend Snippet: Tomato fruit colour DSB repair assay. ( a ) Crossing yellow flesh e 3756 35S:Cas9 and bicolor cc383 u6-26:Ps#1-sgRNA gives F 1 plants with a pale Bicolour fruit phenotype. F 1 plants expressing both Cas9 and gRNA were selected. The gRNA was designed for DSB induction (black lightning) in both alleles between the yellow flesh e 3756 and bicolor cc383 mutations (*). In case of error-prone NHEJ repair (blue line) of bicolor cc383 , fruit colour is yellow. In cases of non-crossover or crossover, fruit colour is expected to be red or bicolour or yellow with red spots in case of late event. Note that whole red fruits were not obtained. Rather, fruits with red spots in a yellow or bicolour background were found and are shown together with additional products of HR-induced repair in Supplementary Fig. 1 . ( b ) Fruit phenotype distribution in F 1 plants and control: Bicolour fruits are shown as orange boxes; Yellow fruits as yellow; Fruits with red sectors (putative somatic HR) are shown as red-stripped boxes. Each column represents a fruit population derived from cross of independent transgenic lines of Cas9 and a given u6-26:Ps#1-sgRNA line. The number of fruits analysed is shown on the column in black. ( c ) Sequences of the NHEJ DSB repair footprints and their relative frequency are shown. The CRISPR-Cas target sequence from the PSY1 is shown on the top. The PSY1 start codon is shown in red and the PAM in blue. The top pie represents an average of illumina Hiseq reads from 22 different F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA. The low pie represents an average of ilummina Hiseq reads from two plants of control F 1 population ( yellow flesh e 3756 × bicolor cc383 F 1 plants with no CRISPR-Cas). ( d ) Inverse PCR scheme for identification of recombinant DNA fragments (details in Supplementary Fig. 4 ). (1) DNA from separate leaves was digested with ApaI(A) and HindIII(H) and then blunted. (2) Each sample was self-ligated, and (3) amplified by two different primer sets (green and yellow). Blue- Bicolor ; red- Yellow flesh ; Dashed blue line- Bicolor deletion, *- Yellow flesh mutation. ( e ) Ratio of parental (P) versus recombinant (R) types (obtained from panel C) in individual plants. Plants 1–15- F 1 plants of the cross of yellow flesh e 3756 35S:Cas9 × bicolor cc383 u6-26:Ps#1-sgRNA; Plant 16- synthetic crossover control; Plants17–18-Yellow flesh × Bicolor (Cas9-) F 1 plants.

    Techniques Used: Expressing, Non-Homologous End Joining, Derivative Assay, Transgenic Assay, CRISPR, Sequencing, Inverse PCR, Recombinant, Amplification, Mutagenesis

    3) Product Images from "Hydrodynamic Delivery of FGF21 Gene Alleviates Obesity and Fatty Liver in Mice Fed a High-fat Diet"

    Article Title: Hydrodynamic Delivery of FGF21 Gene Alleviates Obesity and Fatty Liver in Mice Fed a High-fat Diet

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    doi: 10.1016/j.jconrel.2014.03.047

    Hydrodynamic tail veil injection of plasmid DNA containing FGF21 gene increased FGF21 mRNA levels in the liver, and generated a sustained protein level of FGF21 in the circulation
    Figure Legend Snippet: Hydrodynamic tail veil injection of plasmid DNA containing FGF21 gene increased FGF21 mRNA levels in the liver, and generated a sustained protein level of FGF21 in the circulation

    Techniques Used: Injection, Plasmid Preparation, Generated

    4) Product Images from "A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii"

    Article Title: A nutrient-regulated, dual localization phospholipase A2 in the symbiotic fungus Tuber borchii

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.18.5079

    Fig. 1. TbSP1 identification. ( A ) Equal amounts of SLM-released protein were subjected to SDS–PAGE and stained with Coomassie Blue R-250. Days of in vitro culture (d) and the migration positions of molecular mass markers are indicated at the top and at the left, respectively. The migration position (p23) and the N-terminal sequence (p23/20) of the TbSP1 polypeptide are shown on the right. ( B ) Genomic DNA digested with Eco RI (lane 1), Bam HI (lane 2) or Hin dIII (lane 3) was probed with the TbSP1 cDNA. The migration positions of DNA size markers are indicated on the left. ( C ) Balanced amounts of total RNA extracted from synthetic solid (SSM) or liquid (SLM) mycelial cultures were gel fractionated and probed with the 32 P-labeled TbSP1 cDNA. The migration positions and the amounts of the 28S and 18S rRNAs, utilized as internal references, are shown on the right and in the lower panel, respectively.
    Figure Legend Snippet: Fig. 1. TbSP1 identification. ( A ) Equal amounts of SLM-released protein were subjected to SDS–PAGE and stained with Coomassie Blue R-250. Days of in vitro culture (d) and the migration positions of molecular mass markers are indicated at the top and at the left, respectively. The migration position (p23) and the N-terminal sequence (p23/20) of the TbSP1 polypeptide are shown on the right. ( B ) Genomic DNA digested with Eco RI (lane 1), Bam HI (lane 2) or Hin dIII (lane 3) was probed with the TbSP1 cDNA. The migration positions of DNA size markers are indicated on the left. ( C ) Balanced amounts of total RNA extracted from synthetic solid (SSM) or liquid (SLM) mycelial cultures were gel fractionated and probed with the 32 P-labeled TbSP1 cDNA. The migration positions and the amounts of the 28S and 18S rRNAs, utilized as internal references, are shown on the right and in the lower panel, respectively.

    Techniques Used: SDS Page, Staining, In Vitro, Migration, Sequencing, Labeling

    5) Product Images from "Characterization of a single mutation in TraQ in a strain of Escherichia coli partially resistant to Qβ infection"

    Article Title: Characterization of a single mutation in TraQ in a strain of Escherichia coli partially resistant to Qβ infection

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2015.00124

    mRNA of traA gene analysis. (A) Northern hybridization for traA mRNA and 16S rRNA using the total RNA of LKG/pASK-IBA3plus; left, Anc(C)/pASK-IBA3plus; middle, M54(C)/pASK-IBA3plus; right. The upper and lower figures are X-ray films of traA mRNA and 16S rRNA, respectively. The asterisks (*) indicate the three signals (one weak and two strong). (B) RT-PCR for Anc(C)/pASK-IBA3plus to determine the 5′- and 3′-terminal sequences of traA mRNA. Lambda DNA digested with Sty I was used as a molecular size marker; lane M. RT-PCR products for determination of the 5′-terminus. The three bands were designated as (i), (ii), and (iii), respectively; lane 1. RT-PCR products for determination of the 3′-terminus. The two bands were designated as (iv) and (v), respectively; lane 2. (C) Schematic representations of the start and end positions of traA mRNA. The vertical lines represent the positions of 5′- and 3′-terminal sequences of (i)–(v) shown in (B) . The gray and black boxes represent the positions of traA_r2 and traA_f primer annealing sites in RT-PCR. The arrow represents the position of the traA1 probe annealing site for Northern hybridization.
    Figure Legend Snippet: mRNA of traA gene analysis. (A) Northern hybridization for traA mRNA and 16S rRNA using the total RNA of LKG/pASK-IBA3plus; left, Anc(C)/pASK-IBA3plus; middle, M54(C)/pASK-IBA3plus; right. The upper and lower figures are X-ray films of traA mRNA and 16S rRNA, respectively. The asterisks (*) indicate the three signals (one weak and two strong). (B) RT-PCR for Anc(C)/pASK-IBA3plus to determine the 5′- and 3′-terminal sequences of traA mRNA. Lambda DNA digested with Sty I was used as a molecular size marker; lane M. RT-PCR products for determination of the 5′-terminus. The three bands were designated as (i), (ii), and (iii), respectively; lane 1. RT-PCR products for determination of the 3′-terminus. The two bands were designated as (iv) and (v), respectively; lane 2. (C) Schematic representations of the start and end positions of traA mRNA. The vertical lines represent the positions of 5′- and 3′-terminal sequences of (i)–(v) shown in (B) . The gray and black boxes represent the positions of traA_r2 and traA_f primer annealing sites in RT-PCR. The arrow represents the position of the traA1 probe annealing site for Northern hybridization.

    Techniques Used: Northern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction, Lambda DNA Preparation, Marker

    6) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    Journal: Oncology Reports

    doi: 10.3892/or.2014.3309

    UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P
    Figure Legend Snippet: UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    7) Product Images from "Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors"

    Article Title: Mutations in the Non-Structural Protein-Coding Sequence of Protoparvovirus H-1PV Enhance the Fitness of the Virus and Show Key Benefits Regarding the Transduction Efficiency of Derived Vectors

    Journal: Viruses

    doi: 10.3390/v10040150

    Accumulation of viral DNA replicative forms in infected or transfected NB-324k cells with the mutants. ( A ) Accumulation of DNA replicative forms in NB-324k cells infected with wt H-1PV and derived mutants. NB-324K cells (1.6 × 10 6 ) were infected (MOI, 1 PFU/cell) with wt H-1PV, H1-PM-I, -II, -III, and –DM and further incubated in the presence of neutralizing antibodies. At 20 h post-infection, cells were harvested and viral DNA replicative forms were purified from cell lysates, separated by agarose gel electrophoresis, and subjected to Southern blotting. The bands corresponding to single-stranded, monomeric, and dimeric replicative forms of viral DNA are indicated as ssDNA, mRF, and dRF, respectively ( A ); ( B ) DNA replicative forms in NB-324k cells transfected with the plasmids pH1 (wt) or mutant derivatives. NB-324k cells (1 × 10 6 cells) were transfected with 6 µg of pH1, pH1-PM-I, pH1-PM-II, pH1-PM-III, and pH1-DM and further incubated with neutralizing antibodies. Cells were harvested at indicated times post-transfection and viral DNA replicative forms purified from cell lysates were DpnI digested and analyzed by Southern blotting.
    Figure Legend Snippet: Accumulation of viral DNA replicative forms in infected or transfected NB-324k cells with the mutants. ( A ) Accumulation of DNA replicative forms in NB-324k cells infected with wt H-1PV and derived mutants. NB-324K cells (1.6 × 10 6 ) were infected (MOI, 1 PFU/cell) with wt H-1PV, H1-PM-I, -II, -III, and –DM and further incubated in the presence of neutralizing antibodies. At 20 h post-infection, cells were harvested and viral DNA replicative forms were purified from cell lysates, separated by agarose gel electrophoresis, and subjected to Southern blotting. The bands corresponding to single-stranded, monomeric, and dimeric replicative forms of viral DNA are indicated as ssDNA, mRF, and dRF, respectively ( A ); ( B ) DNA replicative forms in NB-324k cells transfected with the plasmids pH1 (wt) or mutant derivatives. NB-324k cells (1 × 10 6 cells) were transfected with 6 µg of pH1, pH1-PM-I, pH1-PM-II, pH1-PM-III, and pH1-DM and further incubated with neutralizing antibodies. Cells were harvested at indicated times post-transfection and viral DNA replicative forms purified from cell lysates were DpnI digested and analyzed by Southern blotting.

    Techniques Used: Infection, Transfection, Derivative Assay, Incubation, Purification, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis

    8) Product Images from "Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation"

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1295

    Specific and non-specific binding of DNA by ParB: a speculative model for spreading at parS sites. ( A ) A region of a DNA molecule containing a specific binding site is shown. ( B ) Specific binding. At low concentrations, ParB binds to parS sequences via the central helix-turn-helix motifs to form a ParB 2 :DNA complex (supporting data in Figures 1 , 3 and 4 ). ( C ) Non-specific DNA binding. Elevated concentrations of ParB allow co-operative non-specific binding via a second (hypothetical) DNA binding domain (supporting data in Figures 1 and 2 ). The continued self-association of ParB (indicated with arrows) via at least two interfaces subsequently leads to formation of higher order networks and DNA condensation. This transition is not dependent on the presence of parS . ( D ) The condensed nucleoprotein network (supporting data in Figures 5 – 8 ) may contain both specific and non-specific DNA binding sites (see main text for justification) that trap loops of DNA that are anchored around parS if the parS site is present. For simplicity, the specific binding sites for most of the ParB dimers are shown unoccupied. Such structures might bridge larger distances, including between distant parS loci, through the sharing of segments of DNA, or via additional protein:protein interactions (indicated with the faded nucleoprotein complex).
    Figure Legend Snippet: Specific and non-specific binding of DNA by ParB: a speculative model for spreading at parS sites. ( A ) A region of a DNA molecule containing a specific binding site is shown. ( B ) Specific binding. At low concentrations, ParB binds to parS sequences via the central helix-turn-helix motifs to form a ParB 2 :DNA complex (supporting data in Figures 1 , 3 and 4 ). ( C ) Non-specific DNA binding. Elevated concentrations of ParB allow co-operative non-specific binding via a second (hypothetical) DNA binding domain (supporting data in Figures 1 and 2 ). The continued self-association of ParB (indicated with arrows) via at least two interfaces subsequently leads to formation of higher order networks and DNA condensation. This transition is not dependent on the presence of parS . ( D ) The condensed nucleoprotein network (supporting data in Figures 5 – 8 ) may contain both specific and non-specific DNA binding sites (see main text for justification) that trap loops of DNA that are anchored around parS if the parS site is present. For simplicity, the specific binding sites for most of the ParB dimers are shown unoccupied. Such structures might bridge larger distances, including between distant parS loci, through the sharing of segments of DNA, or via additional protein:protein interactions (indicated with the faded nucleoprotein complex).

    Techniques Used: Binding Assay

    Specific binding of ParB to the parS sequence. Electrophoretic mobility shift assay of ParB binding to a radiolabelled 147-bp substrate in a magnesium acetate containing gel-running buffer. ( A ) Titration of ParB on DNA containing a single parS site in the centre. ( B ) ParB titration on an equivalent substrate that is lacking a parS site (see Supplementary Table S1 for details). The species assigned as specific and non-specific complexes are labelled. The lower panels show the quantification of the gels revealing a highly sigmoidal pattern for non-specific binding. These data were fit to Equation ( 1 ) to yield the values shown.
    Figure Legend Snippet: Specific binding of ParB to the parS sequence. Electrophoretic mobility shift assay of ParB binding to a radiolabelled 147-bp substrate in a magnesium acetate containing gel-running buffer. ( A ) Titration of ParB on DNA containing a single parS site in the centre. ( B ) ParB titration on an equivalent substrate that is lacking a parS site (see Supplementary Table S1 for details). The species assigned as specific and non-specific complexes are labelled. The lower panels show the quantification of the gels revealing a highly sigmoidal pattern for non-specific binding. These data were fit to Equation ( 1 ) to yield the values shown.

    Techniques Used: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Titration

    Specific binding of parS to ParB protects the helix-turn-helix region from proteolysis. ParB (2-μM dimer) was progressively digested into a large and a small fragment by trypsin, with approximate weights of 26 and 15 kDa, respectively, as determined by a comparison to molecular weight markers. N-terminal sequencing of the excised bands revealed the N-terminal sequences of these fragments to be MAKX and KXIN, respectively. The N-terminus of the large fragment is M1, with the C-terminus lying within the linker region between the central and C-terminal domains of ParB. The N-terminus of the small fragment is K7, which lies within the Box I motif, and the C-terminus is within the helix-turn-helix motif (K132 or K143). The lower panel shows a cartoon representation of the primary structure indicating the major degradation products. In the presence of parS DNA (20 μM), the degradation of the large fragment to the small fragment (and therefore cleavage near the helix-turn-helix motif) is substantially reduced, whereas an equivalent non-specific DNA does not have this effect.
    Figure Legend Snippet: Specific binding of parS to ParB protects the helix-turn-helix region from proteolysis. ParB (2-μM dimer) was progressively digested into a large and a small fragment by trypsin, with approximate weights of 26 and 15 kDa, respectively, as determined by a comparison to molecular weight markers. N-terminal sequencing of the excised bands revealed the N-terminal sequences of these fragments to be MAKX and KXIN, respectively. The N-terminus of the large fragment is M1, with the C-terminus lying within the linker region between the central and C-terminal domains of ParB. The N-terminus of the small fragment is K7, which lies within the Box I motif, and the C-terminus is within the helix-turn-helix motif (K132 or K143). The lower panel shows a cartoon representation of the primary structure indicating the major degradation products. In the presence of parS DNA (20 μM), the degradation of the large fragment to the small fragment (and therefore cleavage near the helix-turn-helix motif) is substantially reduced, whereas an equivalent non-specific DNA does not have this effect.

    Techniques Used: Binding Assay, Molecular Weight, Sequencing

    Condensation of DNA by ParB monitored by magnetic tweezers. ( A ) Experimental configuration used to measure condensation dynamics mediated by ParB proteins with magnetic tweezers. ( B ) Schematic representation of the parS DNA substrate. ( C ) Condensation assay. At 4-pN stretching force, 1-μM ParB 2 was injected in the fluid cell and incubated for 2 min. Following incubation, the force was reduced to 0.34 pN. In the absence of protein this leads to the change in extension represented in the grey trace. However, in the presence of ParB we observed a progressive decrease of the extension until reaching a final extension near the surface. Raw data were acquired at 60 Hz (red) and filtered down to 2.4 Hz (black).
    Figure Legend Snippet: Condensation of DNA by ParB monitored by magnetic tweezers. ( A ) Experimental configuration used to measure condensation dynamics mediated by ParB proteins with magnetic tweezers. ( B ) Schematic representation of the parS DNA substrate. ( C ) Condensation assay. At 4-pN stretching force, 1-μM ParB 2 was injected in the fluid cell and incubated for 2 min. Following incubation, the force was reduced to 0.34 pN. In the absence of protein this leads to the change in extension represented in the grey trace. However, in the presence of ParB we observed a progressive decrease of the extension until reaching a final extension near the surface. Raw data were acquired at 60 Hz (red) and filtered down to 2.4 Hz (black).

    Techniques Used: Injection, Incubation

    The stoichiometry of the ParB– parS complex. ( A ) Binding of ParB 2 (9 μM) to 24-bp Hex-labelled DNA (10 μM) analysed by SEC-MALS. Only DNA-containing species were observed by monitoring the (normalized) absorbance at 535 nm. With a parS containing DNA substrate (solid line) the major complex has a calculated Mw of 81.6 ± 1.9 kDa, consistent with a single ParB dimer bound to DNA. A lower abundance species is also seen with a calculated Mw of 112.1 ± 3. In contrast, ParB is unable to bind a non-specific substrate (dotted line). In that case, the DNA is found in a late eluting peak, for which no weight could be assigned due to poor light scattering. ( B ) Native-mass spectrometry of ParB binding to a 100-bp substrate containing a single parS sequence predominantly showed a single dimer bound to the DNA, as well as free DNA. The peak assignments are indicated using cartoons on the graph. Binding of ParB to non-specific DNA was not observed.
    Figure Legend Snippet: The stoichiometry of the ParB– parS complex. ( A ) Binding of ParB 2 (9 μM) to 24-bp Hex-labelled DNA (10 μM) analysed by SEC-MALS. Only DNA-containing species were observed by monitoring the (normalized) absorbance at 535 nm. With a parS containing DNA substrate (solid line) the major complex has a calculated Mw of 81.6 ± 1.9 kDa, consistent with a single ParB dimer bound to DNA. A lower abundance species is also seen with a calculated Mw of 112.1 ± 3. In contrast, ParB is unable to bind a non-specific substrate (dotted line). In that case, the DNA is found in a late eluting peak, for which no weight could be assigned due to poor light scattering. ( B ) Native-mass spectrometry of ParB binding to a 100-bp substrate containing a single parS sequence predominantly showed a single dimer bound to the DNA, as well as free DNA. The peak assignments are indicated using cartoons on the graph. Binding of ParB to non-specific DNA was not observed.

    Techniques Used: Binding Assay, Size-exclusion Chromatography, Mass Spectrometry, Sequencing

    The ParB– parS complex does not recruit additional ParB molecules to neighbouring non-specific DNA. The binding of ParB to a 147-bp DNA labelled with Cy3 (Supplementary Table S1) results in an increase in fluorescence intensity. ( A ) Titration of ParB dimer on DNA containing a parS site in its centre. ( B ) Titration on an equivalent substrate that is lacking the parS site. These data were fit to Equation ( 1 ) to yield the values shown. The error bars represent the standard errors from three independent experiments.
    Figure Legend Snippet: The ParB– parS complex does not recruit additional ParB molecules to neighbouring non-specific DNA. The binding of ParB to a 147-bp DNA labelled with Cy3 (Supplementary Table S1) results in an increase in fluorescence intensity. ( A ) Titration of ParB dimer on DNA containing a parS site in its centre. ( B ) Titration on an equivalent substrate that is lacking the parS site. These data were fit to Equation ( 1 ) to yield the values shown. The error bars represent the standard errors from three independent experiments.

    Techniques Used: Binding Assay, Fluorescence, Titration

    ParB-dependent condensation of DNA is reversible. ( A ) Decondensation of DNA by force. Characteristic force-induced decondensation traces for parS substrates are characterized by multiple small steps and a gradual increase of extension. ( B ) Decondensation of DNA by parS competitor DNA. Following condensation by reduction of force, a 5-μM parS competitor DNA was injected into the flow cell resulting in a process of decondensation characterized by large discrete steps. Decondensation stopped at the extension expected for 0.34 pN applied force in the absence of protein (indicated by the grey dashed line). The lack of protein bound to DNA was checked by raising the force up to 4 pN and reduction down to 0 pN; no (de)condensation effects were observed. ( C ) Condensation force dependency on ParB concentration. A maximum condensation force of 2.1 pN was measured at saturating protein concentration for both parS and non-specific DNA substrates. Errors are the standard deviation of measurements on different molecules ( N ≥ 5 molecules). ( D ) Mean force-extension curve of DNA molecules in the presence of 1-μM ParB 2 (circles). The solid line is included as a guide for the eye. Data in squares are the control experiment in the absence of protein and the solid line is a fit to the worm-like chain model. Errors are the standard deviation of measurements on different molecules ( N ≥ 15 molecules).
    Figure Legend Snippet: ParB-dependent condensation of DNA is reversible. ( A ) Decondensation of DNA by force. Characteristic force-induced decondensation traces for parS substrates are characterized by multiple small steps and a gradual increase of extension. ( B ) Decondensation of DNA by parS competitor DNA. Following condensation by reduction of force, a 5-μM parS competitor DNA was injected into the flow cell resulting in a process of decondensation characterized by large discrete steps. Decondensation stopped at the extension expected for 0.34 pN applied force in the absence of protein (indicated by the grey dashed line). The lack of protein bound to DNA was checked by raising the force up to 4 pN and reduction down to 0 pN; no (de)condensation effects were observed. ( C ) Condensation force dependency on ParB concentration. A maximum condensation force of 2.1 pN was measured at saturating protein concentration for both parS and non-specific DNA substrates. Errors are the standard deviation of measurements on different molecules ( N ≥ 5 molecules). ( D ) Mean force-extension curve of DNA molecules in the presence of 1-μM ParB 2 (circles). The solid line is included as a guide for the eye. Data in squares are the control experiment in the absence of protein and the solid line is a fit to the worm-like chain model. Errors are the standard deviation of measurements on different molecules ( N ≥ 15 molecules).

    Techniques Used: Injection, Flow Cytometry, Concentration Assay, Protein Concentration, Standard Deviation

    ParB stabilizes crossovers and writhe formed by DNA braiding and bridging. ( A ) Cartoon of the experiment to braid DNA segments in trans . The application of one turn (clockwise or anti-clockwise) to doubly tethered beads promotes the cross-over of both DNAs, leading to a change of the extension (Δ z ). Subsequent untwisting to zero rotation immediately recovers the original extension. ( B ) Time trace of an experiment with two doubly tethered beads recorded simultaneously on bare DNA. ( C ) In the presence of ParB the cross-over is stabilized, and the extension does not recover after untwisting to zero rotation or even when one additional turn in the direction opposite to the cross-over is applied. ( D ) Injection of 5-μM parS DNA competitor oligonucleotide promotes the recovery of the full extension following ParB-mediated stabilization of a braid. ( E ) Cartoon of the experiment to bridge DNA segments in cis . Single torsionally constrained DNA molecules ( 1 ) are positively supercoiled at 4-pN force by applying 60 turns ( 2 ). Then, 1-μM ParB 2 is injected into the fluid cell ( 3 ). After full exchange of buffer, all of the turns are released ( 4 ). ( F ) DNA extension is displayed as a function of turns to highlight the hysteresis observed due to bridging of different regions of supercoiled DNA after introduction of ParB. The numbers indicate the different stages of the experiment as per the cartoon in part (E).
    Figure Legend Snippet: ParB stabilizes crossovers and writhe formed by DNA braiding and bridging. ( A ) Cartoon of the experiment to braid DNA segments in trans . The application of one turn (clockwise or anti-clockwise) to doubly tethered beads promotes the cross-over of both DNAs, leading to a change of the extension (Δ z ). Subsequent untwisting to zero rotation immediately recovers the original extension. ( B ) Time trace of an experiment with two doubly tethered beads recorded simultaneously on bare DNA. ( C ) In the presence of ParB the cross-over is stabilized, and the extension does not recover after untwisting to zero rotation or even when one additional turn in the direction opposite to the cross-over is applied. ( D ) Injection of 5-μM parS DNA competitor oligonucleotide promotes the recovery of the full extension following ParB-mediated stabilization of a braid. ( E ) Cartoon of the experiment to bridge DNA segments in cis . Single torsionally constrained DNA molecules ( 1 ) are positively supercoiled at 4-pN force by applying 60 turns ( 2 ). Then, 1-μM ParB 2 is injected into the fluid cell ( 3 ). After full exchange of buffer, all of the turns are released ( 4 ). ( F ) DNA extension is displayed as a function of turns to highlight the hysteresis observed due to bridging of different regions of supercoiled DNA after introduction of ParB. The numbers indicate the different stages of the experiment as per the cartoon in part (E).

    Techniques Used: Injection

    ParB-mediated DNA condensation parameters. ( A ) Mean condensation curves for parS (black) and non-specific (red) DNA substrates ( N > 20). ( B ) Distribution of condensation times for parS (black) and non-specific (red) DNA substrates. ( C ) Distribution of final extensions after condensation for parS (black) and non-specific (red) DNA substrates. ( D ) Scatter plot of initial and final extensions for lambda-based substrates (black), parS -based substrates (blue) and pSP73-based substrates (green). All of the data shown were obtained from condensation curves at 0.34 pN.
    Figure Legend Snippet: ParB-mediated DNA condensation parameters. ( A ) Mean condensation curves for parS (black) and non-specific (red) DNA substrates ( N > 20). ( B ) Distribution of condensation times for parS (black) and non-specific (red) DNA substrates. ( C ) Distribution of final extensions after condensation for parS (black) and non-specific (red) DNA substrates. ( D ) Scatter plot of initial and final extensions for lambda-based substrates (black), parS -based substrates (blue) and pSP73-based substrates (green). All of the data shown were obtained from condensation curves at 0.34 pN.

    Techniques Used:

    9) Product Images from "Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer"

    Article Title: Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126501

    An overview of the BEAMing protocol. ( A ) AMELX and AMELY gene products from the preamplification step, ( B ) water-in-oil emulsion droplets that include PCR reaction mix with allele specific primers for AMELX and AMELY genes, a single DNA molecule from the preamplification step, and a single primer-coated magnetic bead, ( C ) oil droplets each harboring either the AMELX or AMELY gene fragment, ( D ) beads coated with either AMELX or AMELY-specific DNA following the BEAMing reaction, ( E ) beads hybridized either with the Cy5-AMELY or FITC-AMELX probe, ( F ) flow cytometry result of a test sample. Green beads represent AMELX, red beads AMELY, and blue beads represent both AMELX and AMELY-specific DNA sequences.
    Figure Legend Snippet: An overview of the BEAMing protocol. ( A ) AMELX and AMELY gene products from the preamplification step, ( B ) water-in-oil emulsion droplets that include PCR reaction mix with allele specific primers for AMELX and AMELY genes, a single DNA molecule from the preamplification step, and a single primer-coated magnetic bead, ( C ) oil droplets each harboring either the AMELX or AMELY gene fragment, ( D ) beads coated with either AMELX or AMELY-specific DNA following the BEAMing reaction, ( E ) beads hybridized either with the Cy5-AMELY or FITC-AMELX probe, ( F ) flow cytometry result of a test sample. Green beads represent AMELX, red beads AMELY, and blue beads represent both AMELX and AMELY-specific DNA sequences.

    Techniques Used: Polymerase Chain Reaction, Flow Cytometry, Cytometry

    10) Product Images from "DNA Barcoding in Nonhuman Primates Reveals Important Limitations in Retrovirus Integration Site Analysis"

    Article Title: DNA Barcoding in Nonhuman Primates Reveals Important Limitations in Retrovirus Integration Site Analysis

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1016/j.omtm.2020.03.021

    Experimental Outline and Clonal Tracking Methods Treatment schematic for pigtail macaques: (1) Animals were “primed” with daily G-CSF and SCF administration to stimulate CD34 + cell production in the bone marrow. (2) CD34 + cells were purified from harvested bone marrow by immunomagnetic bead-based separation. (3) CD34 + hematopoietic stem and progenitor cells (HSPCs) were cultured overnight in HSPC-supportive media with recombinant human growth factors (rhGFs). (4) Cells were transduced with a SIN LV encoding two transgenes, GFP and MGMT ( P140K ), and a 20-bp DNA barcode flanked by primer seeding sequences. The DNA-barcoded LV vector library was validated by next-generation (Next-Gen) sequencing to contain ~0.9 million different barcodes. (5) During transduction cultures each animal received 1,020 cGy total body irradiation (TBI). (6) Approximately 24 h after TBI was completed, transduced cells were washed and formulated in saline containing autologous serum for intravenous infusion. (7) Peripheral blood (PB) and bone marrow (BM) were collected at various time points after transplant for clonal analysis. Red blood cells were removed by ammonium chloride lysis, and resulting white blood cell populations were either submitted in bulk or were further sort purified by either density gradient centrifugation, fluorescence activated cell sorting, or by immunomagnetic bead-based methods. gDNA was extracted from resulting cell populations and subjected to either ISA or DBS. (8) At 1 year after transplant, animals were treated with chemotherapy to induce selection in favor of gene-modified blood cells.
    Figure Legend Snippet: Experimental Outline and Clonal Tracking Methods Treatment schematic for pigtail macaques: (1) Animals were “primed” with daily G-CSF and SCF administration to stimulate CD34 + cell production in the bone marrow. (2) CD34 + cells were purified from harvested bone marrow by immunomagnetic bead-based separation. (3) CD34 + hematopoietic stem and progenitor cells (HSPCs) were cultured overnight in HSPC-supportive media with recombinant human growth factors (rhGFs). (4) Cells were transduced with a SIN LV encoding two transgenes, GFP and MGMT ( P140K ), and a 20-bp DNA barcode flanked by primer seeding sequences. The DNA-barcoded LV vector library was validated by next-generation (Next-Gen) sequencing to contain ~0.9 million different barcodes. (5) During transduction cultures each animal received 1,020 cGy total body irradiation (TBI). (6) Approximately 24 h after TBI was completed, transduced cells were washed and formulated in saline containing autologous serum for intravenous infusion. (7) Peripheral blood (PB) and bone marrow (BM) were collected at various time points after transplant for clonal analysis. Red blood cells were removed by ammonium chloride lysis, and resulting white blood cell populations were either submitted in bulk or were further sort purified by either density gradient centrifugation, fluorescence activated cell sorting, or by immunomagnetic bead-based methods. gDNA was extracted from resulting cell populations and subjected to either ISA or DBS. (8) At 1 year after transplant, animals were treated with chemotherapy to induce selection in favor of gene-modified blood cells.

    Techniques Used: Purification, Cell Culture, Recombinant, Transduction, Plasmid Preparation, Sequencing, Irradiation, Lysis, Gradient Centrifugation, Fluorescence, FACS, Selection, Modification

    11) Product Images from "Age- and Disease-Dependent HERV-W Envelope Allelic Variation in Brain: Association with Neuroimmune Gene Expression"

    Article Title: Age- and Disease-Dependent HERV-W Envelope Allelic Variation in Brain: Association with Neuroimmune Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019176

    HERV-W env sequence mapping and detection. The human genome contains several endogenous retroviral envelope sequences similar to ERVWE1 . ( A ) Chromosomal loci (arrows) depicting sequences similar to ERVWE1 (accession No. NM_014590). The genomic location of the ERVWE1 locus on chromosome 7 is indicated with a box. ( B ) Phylogenetic analysis of HERV-W env gene at 12 different putative gene loci; the dendrogram was constructed based on DNA sequence by the Neighbor-Joining method using the MEGA4 program. The tree is rooted to the MSRV prototype env sequence, which was used as an out-group gene sequence. ( C ) Amplification and cloning of the 650 bp region of HERV-W env gene: ( i ) ethidium bromide-stained agarose gel showing HERV-W env gene amplicons JEG and BeWo cell lines, ( ii ) frequency of HERV-W env cDNA sequences cloned from human fetal astrocytes (HFA), neurons (HFN), microglia (HFM) and immortalized cell lines. In primary cells, HERV-W env sequences were derived from the 7q21.2 (ERVWE1) locus as well as other loci, while all HERV-W env sequences from immortalized cell lines appeared to be encoded by 7q21.2. The values in parenthesis indicate percent diversity among clones for each patient relative to ERVWE1. (ND = none detected).
    Figure Legend Snippet: HERV-W env sequence mapping and detection. The human genome contains several endogenous retroviral envelope sequences similar to ERVWE1 . ( A ) Chromosomal loci (arrows) depicting sequences similar to ERVWE1 (accession No. NM_014590). The genomic location of the ERVWE1 locus on chromosome 7 is indicated with a box. ( B ) Phylogenetic analysis of HERV-W env gene at 12 different putative gene loci; the dendrogram was constructed based on DNA sequence by the Neighbor-Joining method using the MEGA4 program. The tree is rooted to the MSRV prototype env sequence, which was used as an out-group gene sequence. ( C ) Amplification and cloning of the 650 bp region of HERV-W env gene: ( i ) ethidium bromide-stained agarose gel showing HERV-W env gene amplicons JEG and BeWo cell lines, ( ii ) frequency of HERV-W env cDNA sequences cloned from human fetal astrocytes (HFA), neurons (HFN), microglia (HFM) and immortalized cell lines. In primary cells, HERV-W env sequences were derived from the 7q21.2 (ERVWE1) locus as well as other loci, while all HERV-W env sequences from immortalized cell lines appeared to be encoded by 7q21.2. The values in parenthesis indicate percent diversity among clones for each patient relative to ERVWE1. (ND = none detected).

    Techniques Used: Sequencing, Construct, Amplification, Clone Assay, Staining, Agarose Gel Electrophoresis, Derivative Assay

    HERV-W env sequence analyses from autopsied brain white matter. ( A ) Chromosomal loci encoding HERV-W env sequences in MS and non-MS samples showed 7q21.2 was the predominant locus in MS and non-MS samples, while placental clones were derived from multiple chromosomal locations. Values in parenthesis indicate percent diversity among clones for each patient relative to ERVWE1. ( B ) Phylogenetic analysis of HERV-W env sequences from MS and non-MS samples, based on DNA sequence by the neighbor-joining method. The tree was rooted to MSRV prototype env sequence, which was used as an out-group gene sequence. ( C ) Correlation between sequence diversity within samples with respect to ERVWE1 and HERV-W env abundance in MS and non-MS samples. (ND = none detected).
    Figure Legend Snippet: HERV-W env sequence analyses from autopsied brain white matter. ( A ) Chromosomal loci encoding HERV-W env sequences in MS and non-MS samples showed 7q21.2 was the predominant locus in MS and non-MS samples, while placental clones were derived from multiple chromosomal locations. Values in parenthesis indicate percent diversity among clones for each patient relative to ERVWE1. ( B ) Phylogenetic analysis of HERV-W env sequences from MS and non-MS samples, based on DNA sequence by the neighbor-joining method. The tree was rooted to MSRV prototype env sequence, which was used as an out-group gene sequence. ( C ) Correlation between sequence diversity within samples with respect to ERVWE1 and HERV-W env abundance in MS and non-MS samples. (ND = none detected).

    Techniques Used: Sequencing, Mass Spectrometry, Clone Assay, Derivative Assay

    12) Product Images from "Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells"

    Article Title: Long non-coding RNA urothelial carcinoma associated 1 induces cell replication by inhibiting BRG1 in 5637 cells

    Journal: Oncology Reports

    doi: 10.3892/or.2014.3309

    UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P
    Figure Legend Snippet: UCA1 blocks recruitment of BRG1 to chromatin. (A) UCA1 does not affect the ATPase activity of BRG1. The kinetics of BRG1-induced ATP hydrolysis were analyzed in the presence or absence of UCA1. (B) ChIP analysis of BRG1 binding to the p21 promoter in 5637-iUCA1. 5637-NC cells were used as the control. Genomic DNA was fixed and immunoprecipitated using anti-BRG1 antibody, with IgG as a negative control. Real-time PCR was performed using a primer set specific to the BRG1-binding site of p21 promoter. Data were normalized to input and are expressed as the means ± SD of three independent experiments. * P

    Techniques Used: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction

    Related Articles

    Sequencing:

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Clone Assay:

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Amplification:

    Article Title: Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses
    Article Snippet: .. This result demonstrated that adequate distribution of sequencing reads between segments were obtained from a DNA library prepared from samples amplified by Phusion DNA polymerase (DNA library), and by whole-RNA library. .. Consistency of Illumina sequencing, and sequencing analysis of A/PR/8/34 and A/California/07/2009 strains Center for Biologics Evaluation and Research (CBER) stock of A/PR/8/34 was kindly provided by Dr. Peter Palese at Mount.

    Article Title: Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
    Article Snippet: .. Such changes were still observed after amplification with Phusion High Fidelity DNA Polymerase, with 3/15 (20%) of single allele clones obtained from HepG2 and Huh-7 DNA showing apparent poly(T) slippage and also four sequence variants observed that were not seen in other clones of the same haplotype ( ). .. Overall, despite this sequence heterogeneity, the polymorphisms confirmed two different haplotypes in HepG2 DNA, consistent with the presence of two alleles of the pre-mir-122 stem-loop region.

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

    DNA Purification:

    Article Title: Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases
    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. .. Synthetic oligodeoxynucleotides, including a target ( X ) and a probe ( P ), were synthesized by Integrated DNA Technologies, Inc. and purified by high-performance liquid chromatography (HPLC).

    Polymerase Chain Reaction:

    Article Title: Variations of five eIF4E genes across cassava accessions exhibiting tolerant and susceptible responses to cassava brown streak disease
    Article Snippet: .. PCR was performed in a 20 μl reaction volume containing 10 unit Phusion DNA polymerase (NEB, Ipswich, MA), 1 μl of 1:5 diluted cDNA template, 1X Phusion PCR buffer, 5 μM each of upstream and downstream primers, and 250 nM dNTP with the following cycling condition: 98°C for 1 minute; 35 cycles of 98°C for 15 seconds, 56°C for 15 seconds, and 72°C for 45 seconds; and finally 72°C for 5 minutes. .. Primers were designed according to five annotated eIF4E transcripts identified in the draft cassava genomic sequence (Manihot esculenta v4.1) published in Phytozome ( http://phytozome.jgi.doe.gov ) in 2013, prior to the availability of the current cassava genome V6.1 ( ).

    Article Title: Fast and Reliable PCR Amplification from Aspergillus fumigatus Spore Suspension Without Traditional DNA Extraction). Fast and reliable PCR amplification from Aspergillus fumigatus spore suspension without traditional DNA extraction
    Article Snippet: .. Successful PCR amplification has also been obtained with a Phusion High‐Fidelity DNA polymerase (New England Biolabs; M0530; see Fig. A). .. This polymerase was tested in the following PCR conditions for PCR products of ∼1.2 kb: 1 cycle at 98°C for 30 s followed by 35 cycles of 98°C for 10 s, 58°C for 20 s, 72°C for 45 s, and finally 1 cycle of 72°C for 5 min. 8 Following the PCR, mix 5 µl of the PCR reaction with 1 µl of 6× DNA loading dye and load the reactions on an agarose gel (Voytas, ).

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    New England Biolabs nebnext high fidelity pcr dna polymerase
    Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well <t>PCR</t> plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library <t>DNA,</t> we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc
    Nebnext High Fidelity Pcr Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 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
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    New England Biolabs q5 dna high fidelity polymerase
    Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and <t>Q5</t> DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).
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    Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Journal: Genome Biology

    Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

    doi: 10.1186/s13059-018-1407-3

    Figure Lengend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Article Snippet: In addition, we used SuperScript III instead of ProtoScript in the RT step and KAPA HiFi DNA polymerase instead of NEBNext High-Fidelity PCR DNA polymerase in the PCR step.

    Techniques: Lysis, Polymerase Chain Reaction, Flow Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay

    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

    Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

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

    Article Title: DNA synthesis from diphosphate substrates by DNA polymerases

    doi: 10.1073/pnas.1712193115

    Figure Lengend Snippet: Deoxynucleoside diphosphate utilization by DNA polymerases. ( A ) A standard PCR with a monophosphate substrate is inhibited, whereas the presence of all four di- or triphosphate nucleotides supports DNA amplification. Thermophilic polymerases Taq, Vent (exo−), and Pfu , as well as B , Deep Vent, and Q5 DNA polymerases, also utilize the diphosphorylated substrates. ( C and D ) Primer-extension reactions on short templates sampled at indicated time points. All reactions were performed with 100 µM dNDPs at 60 °C, except the Bsu reactions, which were performed at 37 °C. Extended sequences are shown alongside of gels and were distinct in C and D . Primer band is indicated with “–P.” Pausing appears mainly before incorporation of dA and dC and is variable among the polymerases. ( E ).

    Article Snippet: Taq DNA polymerase, Phusion High-Fidelity DNA polymerase, DeepVent DNA polymerase, Vent DNA polymerase, Q5 DNA High-Fidelity polymerase, Bst polymerase, Bsu DNA polymerase (large fragment), and ThermoPol Reaction Buffer [1×: 20 mM Tris⋅HCl, 10 mM (NH4 )2 SO4 , 10 mM KCl, 2 mM MgSO4 , 0.1% Triton–X–100, pH 8.8 at 25 °C] were purchased from New England Biolabs.

    Techniques: Polymerase Chain Reaction, Amplification

    Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Journal: Genome Biology

    Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

    doi: 10.1186/s13059-018-1407-3

    Figure Lengend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Article Snippet: In addition, we used SuperScript III instead of ProtoScript in the RT step and KAPA HiFi DNA polymerase instead of NEBNext High-Fidelity PCR DNA polymerase in the PCR step.

    Techniques: Lysis, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay