exonuclease deficient klenow fragment  (New England Biolabs)


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    Structured Review

    New England Biolabs exonuclease deficient klenow fragment
    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 <t>DNA</t> polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - <t>klenow</t> fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.
    Exonuclease Deficient Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach"

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007029

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.
    Figure Legend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Techniques Used: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing

    2) Product Images from "Single-stranded nucleic acid sensing and coacervation by linker histone H1"

    Article Title: Single-stranded nucleic acid sensing and coacervation by linker histone H1

    Journal: bioRxiv

    doi: 10.1101/2021.03.17.435841

    H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.
    Figure Legend Snippet: H1 coalesces around nascent ssDNA. ( A ) Schematic of the combined single-molecule fluorescence and force microscopy. A biotinylated λ-DNA molecule (48.5 kbp) is tethered between two streptavidin-coated polystyrene beads. ( B ) A representative kymograph of Cy3-H1 binding to DNA over time as the inter-bead distance was increased. ( C ) Total H1 signal across the DNA as a function of time for the kymograph shown in (B). ( D ) Distribution of the H1 signal along the DNA at two specific time points (T1 and T2) as indicated by the arrows in (B). ( E ) Cartoon illustrating the distinct binding configurations of H1 on DNA under different tensions. ssDNA is created by force-induced unpeeling. ( F ) Schematic of two-color imaging for simultaneous visualization of H1 and RPA binding to DNA. ( G ) A representative kymograph of Cy3-H1 (green) and AlexaFluor488-RPA (blue) binding to DNA over time as the inter-bead distance was increased. ( H ) Total H1 and RPA signals across the DNA as a function of time for the kymograph shown in (G). ( I ) Distribution of the H1 (green) and RPA (blue) signals along the DNA at a specific time point (T1) as indicated by the arrow in (G). ( J ) Cartoon illustrating that H1 and RPA occupy separate regions of the tethered DNA. H1 coalesces around relaxed ssDNA, whereas RPA binds to ssDNA under tension.

    Techniques Used: Fluorescence, Microscopy, Binding Assay, Imaging, Recombinase Polymerase Amplification

    3) Product Images from "Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance"

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn575

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Figure Legend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    4) Product Images from "Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase"

    Article Title: Replication Fork Activation Is Enabled by a Single-Stranded DNA Gate in CMG Helicase

    Journal: Cell

    doi: 10.1016/j.cell.2019.06.032

    CMG Undergoes Directional Translocation on ssDNA (A) Schematic of the experimental setup. Individual DNA tethers were formed in channels 1–3 separated by laminar flow containing streptavidin-coated beads, biotinylated λ-DNA, and buffer, respectively. They were subsequently moved to orthogonal channels 4 and 5 for protein loading and imaging. The illustration in the zoom-in box was not drawn to scale. (B) (Left) Cartoon and 2D scan of a tethered ssDNA loaded with multiple Cy3-CMGs. (Right) Representative kymographs of CMG movement in the presence of 1 mM ATP and 10 nM Mcm10 under 5 pN of tension. (C) Representative kymographs of Cy3-CMG (green) on ssDNA in the presence of Mcm10 but without ATP. (D) Distribution of CMG translocation rates on ssDNA in the absence (red) and presence (blue) of ATP ( n = 40 and 62, respectively). .
    Figure Legend Snippet: CMG Undergoes Directional Translocation on ssDNA (A) Schematic of the experimental setup. Individual DNA tethers were formed in channels 1–3 separated by laminar flow containing streptavidin-coated beads, biotinylated λ-DNA, and buffer, respectively. They were subsequently moved to orthogonal channels 4 and 5 for protein loading and imaging. The illustration in the zoom-in box was not drawn to scale. (B) (Left) Cartoon and 2D scan of a tethered ssDNA loaded with multiple Cy3-CMGs. (Right) Representative kymographs of CMG movement in the presence of 1 mM ATP and 10 nM Mcm10 under 5 pN of tension. (C) Representative kymographs of Cy3-CMG (green) on ssDNA in the presence of Mcm10 but without ATP. (D) Distribution of CMG translocation rates on ssDNA in the absence (red) and presence (blue) of ATP ( n = 40 and 62, respectively). .

    Techniques Used: Translocation Assay, Imaging

    5) Product Images from "Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities"

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13324-0

    Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .
    Figure Legend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Techniques Used: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

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    New England Biolabs t7 endonuclease i
    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs klenow fragment exo buffer
    Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), <t>Klenow</t> Fragment <t>exo-</t> (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.
    Klenow Fragment Exo Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs klenow fragment
    In vitro replication assay with the increasing concentration of CU1. Single-stranded <t>DNA</t> template pUC19-sinP3 and its complementary Cy5 labelled primer were annealed and then incubated with increasing concentration of the repressor CU1(in μg/ml). DNA polymerase, <t>Klenow</t> fragment (NEB) and 0.25 ​mM dNTP mixture were then added to the reaction mixtures to initiate DNA polymerization. Lane 1: Free primer, Lanes 2–7: Replication product formation by Klenow fragment (NEB) in the presence of an increasing concentration of CU1(in μg/ml).
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs ts k1 dna polymerase
    Phylogenetic tree showing the evolutionary relationship between Ts <t>K1</t> DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Ts K1 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Deletion of MCM10 through CRISPR/Cas9 technology. Notes:  ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: Ablation of MCM10 using CRISPR/Cas9 restrains the growth and migration of esophageal squamous cell carcinoma cells through inhibition of Akt signaling

    doi: 10.2147/OTT.S157025

    Figure Lengend Snippet: Deletion of MCM10 through CRISPR/Cas9 technology. Notes: ( A ) Validation of CRISPR/Cas9-mediated knockout efficiency using a mCherry/GFP reporter construct. EC109 cells were transfected with the Cas9, sgRNAs, and reporter constructs, and mCherry/GFP fluorescence was examined 48 h after transfection. (a) Bright field image of cells. (b) Some cells displayed GFP fluorescence, indicating the presence of CRISPR/Cas9-mediated removal of target sequence. (c) EC109 cells that were transfected with reporter construct showed mCherry fluorescence. (d) Merged image of green and red fluorescence yielded yellow fluorescence. Scale bar = 100 µm. ( B ) T7 endonuclease assay. Different clones derived from EC109 cells transfected with Cas9 and sgRNAs were subjected to PCR amplification of genomic DNA containing sgRNA-1 target site. The size of T7 endonuclease I-digested DNA fragments is indicated on the right. Control, negative control. ( C ) Upper; RT-PCR analysis of MCM10 mRNA expression in different EC109 sublines. Lower; Western blot analysis of MCM10 protein levels. ( D ) Depletion of MCM10 hampers the migration of ESCC cells. In vitro wound-healing assay was performed to assess cell migration capacity. Top; one representative experiment. The percentage of wound closure was determined from three independent experiments. * P

    Article Snippet: After treatment with T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 2 h, the resulting fragments were subjected to 1% agarose gel electrophoresis and stained with ethidium bromide.

    Techniques: CRISPR, Knock-Out, Construct, Transfection, Fluorescence, Sequencing, Clone Assay, Derivative Assay, Polymerase Chain Reaction, Amplification, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Migration, In Vitro, Wound Healing Assay

    Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), Klenow Fragment exo- (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

    doi: 10.3390/s19010077

    Figure Lengend Snippet: Fluorescence intensity of the proposed aptamer sensing with isothermal circular system containing the DNA aptamer (1 μM), signaling probe (1 μM), primer (2.5 μM), dNTPs (2 mM), Klenow Fragment exo- (1 U/μL), and Nt.BbvCI (1 U/μL), and initiated with a different amount of the PDGF-BB sample (a: 0, b: 0.1 ng/mL, c: 1 ng/mL, d: 10 f ng/mL, e: 20 ng/mL, f: 40 ng/mL, g: 60 ng/mL) at different times.

    Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

    Techniques: Fluorescence

    The fluorescence spectrum of the developed sensing system is collected in a blank control sample (curve “a”); in the presence of aptamer DNA, an MB, polymerase, and nicking endonuclease, but without PDGF-BB (curve “b”); in the presence of PDGF-BB, aptamer DNA, and an MB (curve “c”); in the presence of PDGF-BB, aptamer DNA, an MB, and polymerase (curve “d”); and in the presence of PDGF-BB, aptamer DNA, MB, polymerase, and nicking endonuclease Nt.BbvCI (curve “e”). The reaction is in an NEB buffer (pH 7.9) at 37 °C for 2 h containing 1 μM aptamer DNA, 1 μM MB, 2.5 μM primer, 100 ng/mL PDGF-BB, 1 U/μL Klenow Fragment exo-, 2 mM dNTPs, and 1 U/μL Nt.BbvCI.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins

    doi: 10.3390/s19010077

    Figure Lengend Snippet: The fluorescence spectrum of the developed sensing system is collected in a blank control sample (curve “a”); in the presence of aptamer DNA, an MB, polymerase, and nicking endonuclease, but without PDGF-BB (curve “b”); in the presence of PDGF-BB, aptamer DNA, and an MB (curve “c”); in the presence of PDGF-BB, aptamer DNA, an MB, and polymerase (curve “d”); and in the presence of PDGF-BB, aptamer DNA, MB, polymerase, and nicking endonuclease Nt.BbvCI (curve “e”). The reaction is in an NEB buffer (pH 7.9) at 37 °C for 2 h containing 1 μM aptamer DNA, 1 μM MB, 2.5 μM primer, 100 ng/mL PDGF-BB, 1 U/μL Klenow Fragment exo-, 2 mM dNTPs, and 1 U/μL Nt.BbvCI.

    Article Snippet: The deoxynucleotide solution mixture (dNTPs), polymerase Klenow Fragment exo- (10 U/μL) accompanied by 10× Klenow Fragment exo- buffer, and the nicking endonuclease Nt.BbvCI accompanied by 10× New England Biolabs (NEB) buffer were purchased from New England Biolabs Ltd (Beijing, China).

    Techniques: Fluorescence

    In vitro replication assay with the increasing concentration of CU1. Single-stranded DNA template pUC19-sinP3 and its complementary Cy5 labelled primer were annealed and then incubated with increasing concentration of the repressor CU1(in μg/ml). DNA polymerase, Klenow fragment (NEB) and 0.25 ​mM dNTP mixture were then added to the reaction mixtures to initiate DNA polymerization. Lane 1: Free primer, Lanes 2–7: Replication product formation by Klenow fragment (NEB) in the presence of an increasing concentration of CU1(in μg/ml).

    Journal: Current Research in Pharmacology and Drug Discovery

    Article Title: A saponin-polybromophenol antibiotic (CU1) from Cassia fistula Bark Against Multi-Drug Resistant Bacteria Targeting RNA polymerase

    doi: 10.1016/j.crphar.2022.100090

    Figure Lengend Snippet: In vitro replication assay with the increasing concentration of CU1. Single-stranded DNA template pUC19-sinP3 and its complementary Cy5 labelled primer were annealed and then incubated with increasing concentration of the repressor CU1(in μg/ml). DNA polymerase, Klenow fragment (NEB) and 0.25 ​mM dNTP mixture were then added to the reaction mixtures to initiate DNA polymerization. Lane 1: Free primer, Lanes 2–7: Replication product formation by Klenow fragment (NEB) in the presence of an increasing concentration of CU1(in μg/ml).

    Article Snippet: Then, the primer was allowed to be extended by Klenow fragment (E. coli DNA polymerase; NEB) using the annealed single-stranded DNA as template, in presence of increasing concentrations of CU1.

    Techniques: In Vitro, Concentration Assay, Incubation

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Standard Deviation