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    New England Biolabs bst 2 0 dna polymerase
    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa <t>DNA</t> were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.
    Bst 2 0 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst 2 0 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 497 article reviews
    Price from $9.99 to $1999.99
    bst 2 0 dna polymerase - by Bioz Stars, 2020-07
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    99
    New England Biolabs bst 2 0 warmstart dna polymerase
    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 <t>DNA</t> polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).
    Bst 2 0 Warmstart Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 492 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bst 2 0 warmstart dna polymerase/product/New England Biolabs
    Average 99 stars, based on 492 article reviews
    Price from $9.99 to $1999.99
    bst 2 0 warmstart dna polymerase - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Sensitivity of the L . loa RF4-based LAMP assay. Dilutions of genomic L . loa DNA were amplified with the RF4 primer set and Bst 2.0 DNA polymerase in the absence (blue) or presence (red) of the V/DEF additive. Two ul of each dilution was added to LAMP reactions. The average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the concentration of L . loa DNA (ng/ml). All reactions were performed in triplicate. Error bars represent the standard deviation at each point.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: Lamp Assay, Amplification, Concentration Assay, Standard Deviation

    Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Journal: PLoS ONE

    Article Title: Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pone.0139286

    Figure Lengend Snippet: Detection of L . loa DNA in spiked blood samples. A two-fold dilution series of genomic L . loa DNA was prepared using uninfected human whole blood. NTCs only contained uninfected human whole blood. After DNA isolation, two μl of each dilution (or NTC) was used in LAMP reactions containing the V/DEF additive with Bst 2.0 DNA polymerase. For each experiment, all samples were assayed in triplicate. Average threshold times and standard deviations are plotted against ng DNA/ml of elution buffer.

    Article Snippet: LAMP Assays LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and FB, 1.4 mM of each dNTP, 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 10 mM (NH4 )2 SO4 , 8 mM MgSO4 , 0.1% Tween-20 and 8 U of Bst 2.0 DNA Polymerase (New England Biolabs) mixed with one of several template DNAs, or 10 mM Tris-HCl (pH 8), 0.1 mM EDTA for non-template controls (NTC), in a total volume of 25 μl.

    Techniques: DNA Extraction

    Diagram illustrating the BST–DSN reaction process. ( A ) When dsDNA is used as input in a BST–DSN reaction, the nuclease DSN nicks one strand of dsDNA to create a recognition site for BST polymerase which then synthesizes a complement of the opposite DNA strand while displacing the parent strand. The displaced-sense (or anti-sense) DNA strands subsequently can re-hybridize to complementary strands and form daughter dsDNA. Subsequent DSN nicking and BST amplification generated an exponential amplification of daughter dsDNA while progressively reducing the resulting DNA size. ( B ) When single stranded DNA (ssDNA) or long oligonucleotides are used as input in BST–DSN reaction, the ssDNA is first subjected to a TdT reaction in the presence of dATP to generate a poly-A tail on the 3′ end. The unpurified TdT product is then used as input in a BST–DSN reaction in the presence of an anchored-oligo-dT which is extended by BST to create dsDNA as a first step in the reaction.

    Journal: Nucleic Acids Research

    Article Title: A nuclease-polymerase chain reaction enables amplification of probes used for capture-based DNA target enrichment

    doi: 10.1093/nar/gkz870

    Figure Lengend Snippet: Diagram illustrating the BST–DSN reaction process. ( A ) When dsDNA is used as input in a BST–DSN reaction, the nuclease DSN nicks one strand of dsDNA to create a recognition site for BST polymerase which then synthesizes a complement of the opposite DNA strand while displacing the parent strand. The displaced-sense (or anti-sense) DNA strands subsequently can re-hybridize to complementary strands and form daughter dsDNA. Subsequent DSN nicking and BST amplification generated an exponential amplification of daughter dsDNA while progressively reducing the resulting DNA size. ( B ) When single stranded DNA (ssDNA) or long oligonucleotides are used as input in BST–DSN reaction, the ssDNA is first subjected to a TdT reaction in the presence of dATP to generate a poly-A tail on the 3′ end. The unpurified TdT product is then used as input in a BST–DSN reaction in the presence of an anchored-oligo-dT which is extended by BST to create dsDNA as a first step in the reaction.

    Article Snippet: BST–DSN reaction using PCR products as input BST 2.0 DNA polymerase (BST) and DSN were purchased from NEB and Sapphire North America, respectively.

    Techniques: Amplification, Generated

    Using the LAMP assay for detecting N. caninum plasmid DNA containing the Nc-5 region on a SYBRsafe stained agarose gel. a The effects of reaction time. Lanes 1 and 2: 60 min; Lanes 3 and 4: 50 min; Lanes 5 and 6: 40 min. Lanes 2, 4 and 6 represent negative controls for each reaction time. b Optimizing LAMP assay conditions. Lanes 1 and 2: 30 min; Lanes 3 and 4: 20 min. Lanes 2 and 4 are the negative control for each reaction time. c Restriction analysis of N. caninum LAMP products amplified from plasmid DNA containing the Nc-5 region. The digestion products were run on a 3% agarose gel. Lane 1: N. caninum LAMP product; Lane 2: Msp I digestion of N. caninum product (132–218 bp bands, according to predicted size). Lane MM in a , b and c is the HyperLadder II (Bioline) DNA molecular marker

    Journal: Parasites & Vectors

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

    doi: 10.1186/s13071-017-2549-y

    Figure Lengend Snippet: Using the LAMP assay for detecting N. caninum plasmid DNA containing the Nc-5 region on a SYBRsafe stained agarose gel. a The effects of reaction time. Lanes 1 and 2: 60 min; Lanes 3 and 4: 50 min; Lanes 5 and 6: 40 min. Lanes 2, 4 and 6 represent negative controls for each reaction time. b Optimizing LAMP assay conditions. Lanes 1 and 2: 30 min; Lanes 3 and 4: 20 min. Lanes 2 and 4 are the negative control for each reaction time. c Restriction analysis of N. caninum LAMP products amplified from plasmid DNA containing the Nc-5 region. The digestion products were run on a 3% agarose gel. Lane 1: N. caninum LAMP product; Lane 2: Msp I digestion of N. caninum product (132–218 bp bands, according to predicted size). Lane MM in a , b and c is the HyperLadder II (Bioline) DNA molecular marker

    Article Snippet: LAMP reaction Bst 2.0 DNA Polymerase (New England Biolabs, Herts, UK), having strand displacement activity, was used for LAMP assay amplification at 25 μl final reaction volume.

    Techniques: Lamp Assay, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Negative Control, Amplification, Marker

    LAMP and semi-nested PCR Limit of Detection (LoD). a LAMP reaction. b Semi-nested PCR. Ten-fold serial dilutions of plasmid DNA were used in both a and b ; they contained the Nc-5 region for detecting N. caninum by agarose gel electrophoresis analysis. From left to right: Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of 1 ng plasmid DNA containing the cloned N. caninum fragment; Lanes 2–7: 10-fold serial dilutions of N. caninum plasmid DNA (10 −1 to 10 −6 ng); Lane 8: negative control without target DNA. Semi-nested PCR products showed specific amplification of N. caninum , having 10 −5 ng LoD whereas LAMP LoD was 10 −6 ng. LAMP and semi-nested PCR LoD in both c and d using serial dilutions of N.caninum genomic DNA (NC-1 strain) by visualization on an agarose gel. c LAMP reaction. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of N. caninum genomic DNA (50 ng); Lanes 2–7: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lane 8: positive control (plasmid DNA); Lane 9: negative control (no DNA template). d Semi-nested PCR. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: negative control (no DNA template); Lane 2: amplification of N. caninum genomic DNA (50 ng); Lanes 3–8: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lanes 9, 10: positive controls (plasmid DNA)

    Journal: Parasites & Vectors

    Article Title: A novel loop-mediated isothermal amplification-based test for detecting Neospora caninum DNA

    doi: 10.1186/s13071-017-2549-y

    Figure Lengend Snippet: LAMP and semi-nested PCR Limit of Detection (LoD). a LAMP reaction. b Semi-nested PCR. Ten-fold serial dilutions of plasmid DNA were used in both a and b ; they contained the Nc-5 region for detecting N. caninum by agarose gel electrophoresis analysis. From left to right: Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of 1 ng plasmid DNA containing the cloned N. caninum fragment; Lanes 2–7: 10-fold serial dilutions of N. caninum plasmid DNA (10 −1 to 10 −6 ng); Lane 8: negative control without target DNA. Semi-nested PCR products showed specific amplification of N. caninum , having 10 −5 ng LoD whereas LAMP LoD was 10 −6 ng. LAMP and semi-nested PCR LoD in both c and d using serial dilutions of N.caninum genomic DNA (NC-1 strain) by visualization on an agarose gel. c LAMP reaction. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: amplification of N. caninum genomic DNA (50 ng); Lanes 2–7: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lane 8: positive control (plasmid DNA); Lane 9: negative control (no DNA template). d Semi-nested PCR. Lane MM: HyperLadder II (Bioline) DNA molecular marker; Lane 1: negative control (no DNA template); Lane 2: amplification of N. caninum genomic DNA (50 ng); Lanes 3–8: 10-fold serial dilutions (10 −1 to 10 −6 ng); Lanes 9, 10: positive controls (plasmid DNA)

    Article Snippet: LAMP reaction Bst 2.0 DNA Polymerase (New England Biolabs, Herts, UK), having strand displacement activity, was used for LAMP assay amplification at 25 μl final reaction volume.

    Techniques: Nested PCR, Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Amplification, Clone Assay, Negative Control, Positive Control

    Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Journal: Scientific Reports

    Article Title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Ustilago maydis

    doi: 10.1038/s41598-017-13881-4

    Figure Lengend Snippet: Single factor experiment of RealAmp. The influence of each variable on the LAMP reaction was analyzed by the amplification curve ( a ) and the melt peak ( b ). For the Bst DNA polymerase optimization, Bst polymerase quantities were adjusted to 2 U, 4 U, 6

    Article Snippet: This optimized reaction mixture (total, 25 μl) contained 12.5 μl 2 × Bst DNA polymerase (NEB, Ipswich, MA, USA) reaction buffer [1 × containing 1.6 mM dNTPs, 1 M betaine, 4 mM MgSO4 , 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2 SO4 , and 0.1% Triton X-20 (Sigma-Aldrich Inc., Saint Louis, USA), Double Helix Tech.

    Techniques: Amplification

    Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Species-specificity of Hha I LAMP assay. (A) Each curve represents the calculated average of triplicate turbidity curves generated with various genomic DNAs (0. 1 ng) using Bst 2.0 DNA polymerase without loop primers. Turbidity was observed using B. malayi or B. timori DNA. (B) As a positive control, an actin gene fragment was PCR amplified from B. malayi (Bma), D. immitis (Dim), O. volvulus (Ovo), the mosquito Aedes albopictus (Aal), W. bancrofti (Wba), human (Hsa) and B. timori (Bti) DNAs using degenerate primers. Agarose gel showing amplification of a 244 bp fragment of the actin gene. The 100 bp DNA Ladder (New England Biolabs) was used as the molecular weight marker (MWM). Water was used in the non-template controls (NTC) in (A) and (B).

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Generated, Positive Control, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Molecular Weight, Marker

    Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Sensitivity of Hha I LAMP assay. Ten-fold serial dilutions of B. malayi genomic DNA amplified with the Hha I primer set alone (A) or in the presence of loop primers (B) with Bst DNA polymerase, large fragment (wt Bst LF), Bst 2.0 DNA polymerase ( Bst 2.0) and Bst 2.0 WarmStart DNA polymerase ( Bst 2.0 WS). Data points represent the average of three samples and the error bars represent the standard deviation at each point. For each enzyme, the average threshold time, defined as the time at which the change in turbidity over time (dT/dt) reaches a value of 0.1, is plotted against the amount of starting material. (C) UV detection (365 nm) of products generated within 60 minutes using Bst 2.0 in the presence of loop primers and Fluorescent Detection Reagent. The amount of starting material in ng is shown below the photograph. Positive samples fluoresce green while negative samples remain dark.

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Amplification, Standard Deviation, Generated

    Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

    doi: 10.1371/journal.pntd.0001948

    Figure Lengend Snippet: Hha I LAMP assay for the detection of B. malayi infected blood samples. A set of serial dilutions (two-fold) of microfilariae in blood was prepared and DNA was isolated from each dilution. Three experiments were performed using a different but overlapping range of DNA dilutions. One µl of DNA from each dilution was used in LAMP reactions with Bst 2.0 DNA polymerase. Samples from each experimental set-up were performed in triplicate (experiments 1 and 2) or duplicate (experiment 3). Average threshold times and standard deviations were plotted against the approximate number of mf/µl DNA solution.

    Article Snippet: Reaction times were slightly slower using Bst 2.0 WarmStart DNA polymerase regardless of the presence of loop primers ( ).

    Techniques: Lamp Assay, Infection, Isolation

    The control of enzymatic actuators by the translator module. a Schematic illustration (left) and experimental results (right) of controlling a NanoLuc (NL)-based actuator by the translator module. Experiments were performed using 2 nM of αtoσ , 5 nM of the NanoLuc-based actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM α . The opening of the stem-loop structure of the NanoLuc-based actuator was quantified at intervals of 30 min by measuring the BRET ratio between the NanoLuc donor (em. = 458 nm) and FAM acceptor dye (em. = 533 nm). The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand σ was added for the positive controls. Experiments were performed in triplicate and the fraction in opened conformation in normalized units (n.u.) was calculated by subtracting the mean BRET ratio of the positive controls and normalizing to the mean BRET ratio of the negative controls. Error bars and shaded areas represent the standard error of the mean of the experiments. b Schematic illustration (left) and experimental results (right) of the activation of the self-inhibitory TEM1 β -lactamase (β-lac) actuator by the translator module. Experiments were performed using 12 nM βtoξ , 2.5 nM TEM1 β -lactamase/BLIP actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM β . The activity of TEM1 β -lactamase was measured at time = 0 min prior to initiation with β and 30 min after activation of the translator module and was quantified by measuring the hydrolysis rate of fluorogenic substrate CCF2-FA obtained from the linear regime of the fluorescent time traces. The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand ξ was added for the positive controls. Experiments were performed in triplicate and the activity in normalized units (n.u.) was calculated by subtracting the mean hydrolysis rate of the negative controls and normalizing to the mean hydrolysis rate of the positive controls. Error bars and shaded area’s represent the standard error of the mean of the experiments

    Journal: Nature Communications

    Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories

    doi: 10.1038/s41467-017-01127-w

    Figure Lengend Snippet: The control of enzymatic actuators by the translator module. a Schematic illustration (left) and experimental results (right) of controlling a NanoLuc (NL)-based actuator by the translator module. Experiments were performed using 2 nM of αtoσ , 5 nM of the NanoLuc-based actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM α . The opening of the stem-loop structure of the NanoLuc-based actuator was quantified at intervals of 30 min by measuring the BRET ratio between the NanoLuc donor (em. = 458 nm) and FAM acceptor dye (em. = 533 nm). The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand σ was added for the positive controls. Experiments were performed in triplicate and the fraction in opened conformation in normalized units (n.u.) was calculated by subtracting the mean BRET ratio of the positive controls and normalizing to the mean BRET ratio of the negative controls. Error bars and shaded areas represent the standard error of the mean of the experiments. b Schematic illustration (left) and experimental results (right) of the activation of the self-inhibitory TEM1 β -lactamase (β-lac) actuator by the translator module. Experiments were performed using 12 nM βtoξ , 2.5 nM TEM1 β -lactamase/BLIP actuator, 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and initiated with 30 nM β . The activity of TEM1 β -lactamase was measured at time = 0 min prior to initiation with β and 30 min after activation of the translator module and was quantified by measuring the hydrolysis rate of fluorogenic substrate CCF2-FA obtained from the linear regime of the fluorescent time traces. The translator module was omitted for negative (−) and positive (+) controls and excess of DNA strand ξ was added for the positive controls. Experiments were performed in triplicate and the activity in normalized units (n.u.) was calculated by subtracting the mean hydrolysis rate of the negative controls and normalizing to the mean hydrolysis rate of the positive controls. Error bars and shaded area’s represent the standard error of the mean of the experiments

    Article Snippet: 2.0 WarmStart DNA polymerase were obtained from NEB, while ttRecJ, a thermophilic equivalent of the RecJ enzyme from Thermus thermophilus , was obtained from André Estévez-Torres.

    Techniques: Bioluminescence Resonance Energy Transfer, Activation Assay, Activity Assay

    Control of two orthogonal enzymatic actuators by a switchable memories circuit. a Schematics of the experiment in which the switch controls a NanoLuc-based actuator and a self-inhibitory TEM1 β -lactamase construct. b – d Results of the experiments, carried out as described in the Methods, using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. Reactions were performed in presence of the actuators or MBs. The switch was initiated with 1 nM α . b The graphs show the dynamics of the switch and the production of σ and ξ measured using MBs (5 nM MB σ and 2.5 nM MB ξ ) (Supplementary Fig. 17 ) in absence (light color) and in presence (dark color) of the translator modules (2 nM αtoσ and 12 nM βtoξ ). The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of β and α , respectively. The dotted lines show the time points at which 30 nM of the Inputs δ and γ were added. In parallel, experiments were run where the MBs were replaced with the enzymatic actuators (5 nM of the NanoLuc-based actuator and 2.5 nM TEM1 β -lactamase actuator). c , d The state of the actuators was measured at four time points including negative (−) and positive (+) controls (Supplementary Figs. 13 , 14 and Methods). Error bars and shaded area’s represent the standard error of the mean of the experiments. Experiments were performed in plurality ( > 3) and at three different days. c The bar graphs displaying the BRET ratio were normalized to the mean of the negative controls for a clear visualization (Supplementary Fig. 14 displays the raw data). d The activity or fraction in opened conformation of the actuators were calculated by normalizing to positive and negative controls (Methods)

    Journal: Nature Communications

    Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories

    doi: 10.1038/s41467-017-01127-w

    Figure Lengend Snippet: Control of two orthogonal enzymatic actuators by a switchable memories circuit. a Schematics of the experiment in which the switch controls a NanoLuc-based actuator and a self-inhibitory TEM1 β -lactamase construct. b – d Results of the experiments, carried out as described in the Methods, using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. Reactions were performed in presence of the actuators or MBs. The switch was initiated with 1 nM α . b The graphs show the dynamics of the switch and the production of σ and ξ measured using MBs (5 nM MB σ and 2.5 nM MB ξ ) (Supplementary Fig. 17 ) in absence (light color) and in presence (dark color) of the translator modules (2 nM αtoσ and 12 nM βtoξ ). The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of β and α , respectively. The dotted lines show the time points at which 30 nM of the Inputs δ and γ were added. In parallel, experiments were run where the MBs were replaced with the enzymatic actuators (5 nM of the NanoLuc-based actuator and 2.5 nM TEM1 β -lactamase actuator). c , d The state of the actuators was measured at four time points including negative (−) and positive (+) controls (Supplementary Figs. 13 , 14 and Methods). Error bars and shaded area’s represent the standard error of the mean of the experiments. Experiments were performed in plurality ( > 3) and at three different days. c The bar graphs displaying the BRET ratio were normalized to the mean of the negative controls for a clear visualization (Supplementary Fig. 14 displays the raw data). d The activity or fraction in opened conformation of the actuators were calculated by normalizing to positive and negative controls (Methods)

    Article Snippet: 2.0 WarmStart DNA polymerase were obtained from NEB, while ttRecJ, a thermophilic equivalent of the RecJ enzyme from Thermus thermophilus , was obtained from André Estévez-Torres.

    Techniques: Construct, Fluorescence, Bioluminescence Resonance Energy Transfer, Activity Assay

    Characterizing retroactivity from coupling of the translator module to the memories circuit. a Schematic illustration of the system, in which the translator module is coupled to α or β of the PEN-based bistable switch. The core of the bistable switch consists of four templates, including the autocatalytic templates αtoα and βtoβ and the inhibitory templates αtoiβ and βtoiα . The network switches between states upon injection of γ and δ which are received by templates γtoα and δtoβ . The dynamics of the bistable switch are followed via N-quenching using templates βtoiα and αtoiβ which are 3′-end labeled with a DY530 and FAM fluorophore, respectively. b Experimental (Exp.) and simulated (Sim.) phase diagrams for a concentration range of translator template coupled to α or β . Experiments were carried out as described in the Methods using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. The switch was either equilibrated to its α-state and 30 nM δ was injected for switching to the β-state or the switch was equilibrated to its β-state and 30 nM γ was injected for switching to the α-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of primer β and α , respectively. The blue and green circles represent the α-state and β-state, respectively. Simulations were performed using the heuristic model ( Supplementary Notes ) and the traces were converted to normalized units (n.u.) by normalizing α and β to their steady-state concentrations. c Bifurcation diagrams of the switch in isolation and with 10 nM of translator module coupled to β or α as a function of inputs γ and δ obtained using the heuristic model ( Supplementary Notes ). The monostable domains of α and β are shown in blue and green, respectively, while the bistable domain is shown in purple

    Journal: Nature Communications

    Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories

    doi: 10.1038/s41467-017-01127-w

    Figure Lengend Snippet: Characterizing retroactivity from coupling of the translator module to the memories circuit. a Schematic illustration of the system, in which the translator module is coupled to α or β of the PEN-based bistable switch. The core of the bistable switch consists of four templates, including the autocatalytic templates αtoα and βtoβ and the inhibitory templates αtoiβ and βtoiα . The network switches between states upon injection of γ and δ which are received by templates γtoα and δtoβ . The dynamics of the bistable switch are followed via N-quenching using templates βtoiα and αtoiβ which are 3′-end labeled with a DY530 and FAM fluorophore, respectively. b Experimental (Exp.) and simulated (Sim.) phase diagrams for a concentration range of translator template coupled to α or β . Experiments were carried out as described in the Methods using 20 nM βtoiα , 15 nM αtoiβ , 24 nM βtoβ , 10 nM αtoα , γtoα , and δtoβ , 15 U mL −1 Bst 2.0 WarmStart DNA polymerase, 10 U mL −1 Nt. bstNBI, and 200 nM ttRecJ. The switch was either equilibrated to its α-state and 30 nM δ was injected for switching to the β-state or the switch was equilibrated to its β-state and 30 nM γ was injected for switching to the α-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the steady-state value of primer β and α , respectively. The blue and green circles represent the α-state and β-state, respectively. Simulations were performed using the heuristic model ( Supplementary Notes ) and the traces were converted to normalized units (n.u.) by normalizing α and β to their steady-state concentrations. c Bifurcation diagrams of the switch in isolation and with 10 nM of translator module coupled to β or α as a function of inputs γ and δ obtained using the heuristic model ( Supplementary Notes ). The monostable domains of α and β are shown in blue and green, respectively, while the bistable domain is shown in purple

    Article Snippet: 2.0 WarmStart DNA polymerase were obtained from NEB, while ttRecJ, a thermophilic equivalent of the RecJ enzyme from Thermus thermophilus , was obtained from André Estévez-Torres.

    Techniques: Injection, Labeling, Concentration Assay, Fluorescence, Isolation

    Coupling of the translator module to an upstream INVERTER network. a Schematic illustration of the translator module coupled to a PEN-based INVERTER network. Multiplex monitoring of the dynamics of the network is performed using endogenous template βtoiα and an exogenous template αtoiβ which are 3′-end fluorescently labeled with DY530 and FAM, respectively, while the output strand σ of the translator module is measured via a MB bearing a fluorophore-quencher pair. b Results of the experiments that were conducted for 0, 2, 5, 10, 20, and 40 nM (light to dark) of translator template αtoσ in the presence of 7 nM αtoα , 20 nM of βtoiα and αtoiβ , 30 nM MB, 10 U mL −1 Bst 2.0 WarmStart DNA polymerase, 25 U mL −1 Nt. bstNBI, and 50 nM ttRecJ. The INVERTER is activated by addition of 0.5 nM α which is initially amplified until it reaches steady-state in which production by polymerase and nickase and degradation due to exonuclease are balanced. Applying a pulse of 30 nM of input β at this point initiates the production of iα , which inhibits autocatalytic production of output α . As input strand β gets degraded the system returns its pre-stimulus steady-state. Hence, the INVERTER network shows a pulse response after injection of input β , which can be characterized by its amplitude and response time which is the time needed to recover to the pre-stimulus steady-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the maximal or steady-state value of primer β and α , respectively. The fluorescence of Cy5 fluorophore was converted to concentration of DNA strand σ using a standard curve (Supplementary Fig. 17 ). c Results of simulations using the heuristic model with the same concentrations of translator template as used during the experiments in b and for different values of ρ . The traces were converted to normalized units (n.u.) by normalizing α to the steady-state concentration and normalizing β to its maximum value

    Journal: Nature Communications

    Article Title: Hierarchical control of enzymatic actuators using DNA-based switchable memories

    doi: 10.1038/s41467-017-01127-w

    Figure Lengend Snippet: Coupling of the translator module to an upstream INVERTER network. a Schematic illustration of the translator module coupled to a PEN-based INVERTER network. Multiplex monitoring of the dynamics of the network is performed using endogenous template βtoiα and an exogenous template αtoiβ which are 3′-end fluorescently labeled with DY530 and FAM, respectively, while the output strand σ of the translator module is measured via a MB bearing a fluorophore-quencher pair. b Results of the experiments that were conducted for 0, 2, 5, 10, 20, and 40 nM (light to dark) of translator template αtoσ in the presence of 7 nM αtoα , 20 nM of βtoiα and αtoiβ , 30 nM MB, 10 U mL −1 Bst 2.0 WarmStart DNA polymerase, 25 U mL −1 Nt. bstNBI, and 50 nM ttRecJ. The INVERTER is activated by addition of 0.5 nM α which is initially amplified until it reaches steady-state in which production by polymerase and nickase and degradation due to exonuclease are balanced. Applying a pulse of 30 nM of input β at this point initiates the production of iα , which inhibits autocatalytic production of output α . As input strand β gets degraded the system returns its pre-stimulus steady-state. Hence, the INVERTER network shows a pulse response after injection of input β , which can be characterized by its amplitude and response time which is the time needed to recover to the pre-stimulus steady-state. The charge level is the normalized fluorescence of the signal of DY530 and FAM fluorophores, which is 0 in the absence of template’s input primer and 1 at the maximal or steady-state value of primer β and α , respectively. The fluorescence of Cy5 fluorophore was converted to concentration of DNA strand σ using a standard curve (Supplementary Fig. 17 ). c Results of simulations using the heuristic model with the same concentrations of translator template as used during the experiments in b and for different values of ρ . The traces were converted to normalized units (n.u.) by normalizing α to the steady-state concentration and normalizing β to its maximum value

    Article Snippet: 2.0 WarmStart DNA polymerase were obtained from NEB, while ttRecJ, a thermophilic equivalent of the RecJ enzyme from Thermus thermophilus , was obtained from André Estévez-Torres.

    Techniques: Multiplex Assay, Labeling, Amplification, Injection, Fluorescence, Concentration Assay

    Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Journal: MethodsX

    Article Title: NGS-based deep bisulfite sequencing

    doi: 10.1016/j.mex.2015.11.008

    Figure Lengend Snippet: Overall scheme for NGS-based deep bisulfite sequencing. (A) The entire procedure of the NGS-based deep bisulfite sequencing protocol is shown as a flow chart. (B) The adaptor ligation step for the current protocol has adopted one strategy, in which the added PCR products and two adaptors are ligated through two stepwise incubations. Since both adaptors lack the phosphate group at their 5′-ends, the ligation reaction by T4 at 25 °C occurs between only one strand of the adaptors and the PCR products. In this case, the phosphate groups are derived from the 5′-end of the PCR products. At 65 °C, the activated Bst2.0 WarmStart polymerase extends and displaces the other unligated strand from the partially joined products. The * symbol indicates the phosphate group at the 5′-end of the end-repaired PCR products. (C) The sequences of Ion Torrent P1 and A adaptors are shown with different colors to indicate the key, barcode and spacer regions. The * symbol indicates a phosphothiate bonding between two nucleotides, which protects the duplex adaptor from being digested by the exonuclease activity of DNA polymerases.

    Article Snippet: The current protocol also uses a mixture of T4 ligase and Bst2.0 WarmStart polymerase with two stepwise incubations, a 25-min incubation at 25 °C for the ligation reaction by T4 ligase and another 30-min incubation at 65 °C for the extension/displacement reaction by Bst2.0 WarmStart polymerase (available from NEB).

    Techniques: Next-Generation Sequencing, Methylation Sequencing, Ligation, Polymerase Chain Reaction, Derivative Assay, Activity Assay