dna polymerase i  (Thermo Fisher)


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    Name:
    Klenow Fragment
    Description:
    Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I It exhibits 5 →3 polymerase activity and 3 →5 exonuclease proofreading activity but lacks 5 →3 exonuclease activity of DNA polymerase I Highlights• Incorporates modified nucleotides e g Cy3 Cy5 aminoallyl biotin digoxigenin and fluorescently labeled nucleotides • Active in restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• DNA blunting by fill in 5 overhangs• Random primed DNA labeling• Labeling by fill in 5 overhangs of dsDNA• DNA sequencing by the Sanger method• Site specific mutagenesis of DNA with synthetic oligonucleotides• Second strand synthesis of cDNA
    Catalog Number:
    ep0054
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis|DNA Labeling|Nucleic Acid Labeling & Oligo Synthesis|cDNA Libraries & Library Construction|Mutagenesis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher dna polymerase i
    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS <t>DNA</t> synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I It exhibits 5 →3 polymerase activity and 3 →5 exonuclease proofreading activity but lacks 5 →3 exonuclease activity of DNA polymerase I Highlights• Incorporates modified nucleotides e g Cy3 Cy5 aminoallyl biotin digoxigenin and fluorescently labeled nucleotides • Active in restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• DNA blunting by fill in 5 overhangs• Random primed DNA labeling• Labeling by fill in 5 overhangs of dsDNA• DNA sequencing by the Sanger method• Site specific mutagenesis of DNA with synthetic oligonucleotides• Second strand synthesis of cDNA
    https://www.bioz.com/result/dna polymerase i/product/Thermo Fisher
    Average 97 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    dna polymerase i - by Bioz Stars, 2020-07
    97/100 stars

    Images

    1) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2012.11.039

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    2) Product Images from "Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells"

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

    Journal: Nature biotechnology

    doi: 10.1038/nbt.2720

    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.
    Figure Legend Snippet: Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Techniques Used: Amplification, Lysis, Neutralization, Multiple Displacement Amplification, Transferring, Microscopy, Fluorescence, Micromanipulation, Electron Microscopy, Incubation, Evaporation, Sequencing

    3) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2012.11.039

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    4) Product Images from "A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA"

    Article Title: A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0066864

    The detection of nuclear DNA in HeLa cells. The detection of nuclear DNA by DNA polymerase I (A, D) or TdT (B, C, E) and Alexa 555-dUTP after 10-minute incubation in the cleavage solution is shown. In the case of TdT, the incubation with TdT was (B) or was not (C) preceded by SAP treatment to remove the phosphate groups from the 3′ ends. The cells were (A, B, C) or were not (D, E) incubated in the cleavage solution for 10 minutes before the enzymatic detection of DNA. The relative mean signal intensities and the standard deviations are shown in (F) for all these experiments. Barr: 20 µm.
    Figure Legend Snippet: The detection of nuclear DNA in HeLa cells. The detection of nuclear DNA by DNA polymerase I (A, D) or TdT (B, C, E) and Alexa 555-dUTP after 10-minute incubation in the cleavage solution is shown. In the case of TdT, the incubation with TdT was (B) or was not (C) preceded by SAP treatment to remove the phosphate groups from the 3′ ends. The cells were (A, B, C) or were not (D, E) incubated in the cleavage solution for 10 minutes before the enzymatic detection of DNA. The relative mean signal intensities and the standard deviations are shown in (F) for all these experiments. Barr: 20 µm.

    Techniques Used: Incubation

    The detection of overall DNA by various marker nucleosides. The detection of overall DNA by DNA polymerase I and either by Alexa 555-dUTP (A, C, J, L; red in the color images) or biotin-dUTP (D, F, M, O; red in the color images) or BrdUTP (P, R; red in the color images) in HeLa cells after 10-minute incubation in the cleavage solution is shown. The cells were simultaneously stained by DAPI (B, C, E, F, H, I, K, L, N, O, Q, R; blue in the color images). The cells in A–F were incubated in the DNA polymerase I mixture containing dTTP. The cells in J–R were incubated in the DNA polymerase I mixture without the addition of dTTP. The cells in G–I were fixed, permeabilized, incubated with anti-BrdU antibody and secondary antibody, and the cells were stained by DAPI. Bar: 50 µm.
    Figure Legend Snippet: The detection of overall DNA by various marker nucleosides. The detection of overall DNA by DNA polymerase I and either by Alexa 555-dUTP (A, C, J, L; red in the color images) or biotin-dUTP (D, F, M, O; red in the color images) or BrdUTP (P, R; red in the color images) in HeLa cells after 10-minute incubation in the cleavage solution is shown. The cells were simultaneously stained by DAPI (B, C, E, F, H, I, K, L, N, O, Q, R; blue in the color images). The cells in A–F were incubated in the DNA polymerase I mixture containing dTTP. The cells in J–R were incubated in the DNA polymerase I mixture without the addition of dTTP. The cells in G–I were fixed, permeabilized, incubated with anti-BrdU antibody and secondary antibody, and the cells were stained by DAPI. Bar: 50 µm.

    Techniques Used: Marker, Incubation, Staining

    The detection of the mitochondrial genome. The detection of the mitochondrial DNA by a 10-second cleavage in a solution containing copper(I) ions followed by the labeling of mitochondrial DNA by DNA polymerase I and biotin-dUTP (A, D; red in the color image) or Alexa 555-dUTP (F, I; red in the color image). The anti-mitochondrial antibody (B, D, G, I; green in the color images) was used for the identification of mitochondria. DNA was stained by DAPI (C, D, H, I; blue in the color images). The graphs show the relative mean signal intensities and the standard deviations of Alexa 555-dUTP (E) and biotin-dUTP (J) derived signal measured in the nucleus, mitochondria and cytoplasm (background). Bar: 10 µm.
    Figure Legend Snippet: The detection of the mitochondrial genome. The detection of the mitochondrial DNA by a 10-second cleavage in a solution containing copper(I) ions followed by the labeling of mitochondrial DNA by DNA polymerase I and biotin-dUTP (A, D; red in the color image) or Alexa 555-dUTP (F, I; red in the color image). The anti-mitochondrial antibody (B, D, G, I; green in the color images) was used for the identification of mitochondria. DNA was stained by DAPI (C, D, H, I; blue in the color images). The graphs show the relative mean signal intensities and the standard deviations of Alexa 555-dUTP (E) and biotin-dUTP (J) derived signal measured in the nucleus, mitochondria and cytoplasm (background). Bar: 10 µm.

    Techniques Used: Labeling, Staining, Derivative Assay

    The scheme of the method. A simplified scheme of two basic alternatives of labeling cellular double-stranded DNA is shown. The common steps (fixation, permeabilization and copper(I)-mediated DNA cleavage leading to the gap formation) are followed by the labeling of DNA by means of DNA polymerase I or TdT. In the case of TdT, it is necessary to use the pre-incubation step with SAP in order to reconstitute the hydroxyl groups at the 3′ ends of the gaps. P and OH designate phosphate and hydroxyl groups at the 3′ ends of the gaps, respectively.
    Figure Legend Snippet: The scheme of the method. A simplified scheme of two basic alternatives of labeling cellular double-stranded DNA is shown. The common steps (fixation, permeabilization and copper(I)-mediated DNA cleavage leading to the gap formation) are followed by the labeling of DNA by means of DNA polymerase I or TdT. In the case of TdT, it is necessary to use the pre-incubation step with SAP in order to reconstitute the hydroxyl groups at the 3′ ends of the gaps. P and OH designate phosphate and hydroxyl groups at the 3′ ends of the gaps, respectively.

    Techniques Used: Labeling, Incubation

    5) Product Images from "Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood"

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr424

    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.
    Figure Legend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Techniques Used: Activity Assay, Hybridization, Generated

    6) Product Images from "Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ"

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052584

    The gaps detection, the effect of SOD, Hepes and Tris-HCl and the abasic sites detection. A ) The detection of the gaps produced by the incubation of cells with 4 mM copper(I) for 30 minutes or by the incubation of cells in the solution of 4 mM copper(II) sulfate for 5 minutes, followed by the reduction of the DNA-bound bivalent copper ions by 10 mM sodium ascorbate for 5 minutes is shown. The gaps were visualized by means of DNA polymerase I and Alexa-dUTP. Bar: 20 µm. B ) The effect of SOD on plasmid DNA cleavage is shown. The plasmid DNA was incubated in the cleavage solution for 5, 10 and 30 minutes without (lines 1, 2, 3, respectively) or with SOD (lines 4, 5, 6, respectively). Line 7 represents the copper(I) untreated sample; line M represents the DNA molecular mass marker. Only a very low inhibition of DNA cleavage by SOD was observed. C ) The effect of Hepes and Tris-HCl is shown. The plasmid DNA was incubated in cleavage solution for 0, 5, 10 and 30 minutes alone (lines 1, 2, 3, 4, respectively), with 0.2 M Hepes (lines 5, 6, 7, 8, respectively) or with 0.1 M Tris-HCl (lines 9, 10, 11, 12, respectively). Line M represents the DNA molecular mass marker. Only Tris-HCl efficiently blocked DNA cleavage. D ) The effect of piperidine is shown. The plasmid DNA was incubated in cleavage solution for 30 minutes and subsequently in 1 M piperidine (line 1) or distilled water (line 2) at 90°C. Line M represents the DNA molecular mass marker. The shift to the shorter DNA fragments after piperidine treatment indicates formation of abasic sites.
    Figure Legend Snippet: The gaps detection, the effect of SOD, Hepes and Tris-HCl and the abasic sites detection. A ) The detection of the gaps produced by the incubation of cells with 4 mM copper(I) for 30 minutes or by the incubation of cells in the solution of 4 mM copper(II) sulfate for 5 minutes, followed by the reduction of the DNA-bound bivalent copper ions by 10 mM sodium ascorbate for 5 minutes is shown. The gaps were visualized by means of DNA polymerase I and Alexa-dUTP. Bar: 20 µm. B ) The effect of SOD on plasmid DNA cleavage is shown. The plasmid DNA was incubated in the cleavage solution for 5, 10 and 30 minutes without (lines 1, 2, 3, respectively) or with SOD (lines 4, 5, 6, respectively). Line 7 represents the copper(I) untreated sample; line M represents the DNA molecular mass marker. Only a very low inhibition of DNA cleavage by SOD was observed. C ) The effect of Hepes and Tris-HCl is shown. The plasmid DNA was incubated in cleavage solution for 0, 5, 10 and 30 minutes alone (lines 1, 2, 3, 4, respectively), with 0.2 M Hepes (lines 5, 6, 7, 8, respectively) or with 0.1 M Tris-HCl (lines 9, 10, 11, 12, respectively). Line M represents the DNA molecular mass marker. Only Tris-HCl efficiently blocked DNA cleavage. D ) The effect of piperidine is shown. The plasmid DNA was incubated in cleavage solution for 30 minutes and subsequently in 1 M piperidine (line 1) or distilled water (line 2) at 90°C. Line M represents the DNA molecular mass marker. The shift to the shorter DNA fragments after piperidine treatment indicates formation of abasic sites.

    Techniques Used: Produced, Incubation, Plasmid Preparation, Marker, Inhibition

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    7) Product Images from "Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region"

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr259

    Gel shift studies of plasmid DNA bound with PNAs. After incubation with PNAs (TE plus 20 mM KCl, pH 7.4, 37°C, overnight) at varying PNA molar ratios, with (right) or without (left) bis-PNA, plasmids were cut with SpeI and Pst1 restriction enzymes followed by 3′- 32 P labeling with [α- 32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I. After purification on G-50 microcolumns samples were analyzed in native 6% polyacrylamide gel electrophoresis.
    Figure Legend Snippet: Gel shift studies of plasmid DNA bound with PNAs. After incubation with PNAs (TE plus 20 mM KCl, pH 7.4, 37°C, overnight) at varying PNA molar ratios, with (right) or without (left) bis-PNA, plasmids were cut with SpeI and Pst1 restriction enzymes followed by 3′- 32 P labeling with [α- 32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I. After purification on G-50 microcolumns samples were analyzed in native 6% polyacrylamide gel electrophoresis.

    Techniques Used: Electrophoretic Mobility Shift Assay, Plasmid Preparation, Incubation, Labeling, Purification, Polyacrylamide Gel Electrophoresis

    8) Product Images from "BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction"

    Article Title: BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00366

    Schematic diagram of strand-specific library synthesis mechanism . mRNAs are fragmented by heat and magnesium (1) and primed for cDNA synthesis by an adapter-containing oligonucleotide (2,3) . Size selection and cleanup removes unincorperated oligonucleotides and small cDNA fragments (4) . Transient duplex breathing at the terminus of the RNA-cDNA hybrid (5) facilitates interaction with the single-stranded portion of the 5-prime capturing adapter (6) and E. coli DNA Polymerase I catalyses its incorporation into a complete library molecule (7) .
    Figure Legend Snippet: Schematic diagram of strand-specific library synthesis mechanism . mRNAs are fragmented by heat and magnesium (1) and primed for cDNA synthesis by an adapter-containing oligonucleotide (2,3) . Size selection and cleanup removes unincorperated oligonucleotides and small cDNA fragments (4) . Transient duplex breathing at the terminus of the RNA-cDNA hybrid (5) facilitates interaction with the single-stranded portion of the 5-prime capturing adapter (6) and E. coli DNA Polymerase I catalyses its incorporation into a complete library molecule (7) .

    Techniques Used: Selection

    9) Product Images from "Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region"

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr259

    Gel shift studies of plasmid DNA bound with PNAs. After incubation with PNAs (TE plus 20 mM KCl, pH 7.4, 37°C, overnight) at varying PNA molar ratios, with (right) or without (left) bis-PNA, plasmids were cut with SpeI and Pst1 restriction enzymes followed by 3′- 32 P labeling with [α- 32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I. After purification on G-50 microcolumns samples were analyzed in native 6% polyacrylamide gel electrophoresis.
    Figure Legend Snippet: Gel shift studies of plasmid DNA bound with PNAs. After incubation with PNAs (TE plus 20 mM KCl, pH 7.4, 37°C, overnight) at varying PNA molar ratios, with (right) or without (left) bis-PNA, plasmids were cut with SpeI and Pst1 restriction enzymes followed by 3′- 32 P labeling with [α- 32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I. After purification on G-50 microcolumns samples were analyzed in native 6% polyacrylamide gel electrophoresis.

    Techniques Used: Electrophoretic Mobility Shift Assay, Plasmid Preparation, Incubation, Labeling, Purification, Polyacrylamide Gel Electrophoresis

    10) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2012.11.039

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    11) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2012.11.039

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    12) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2012.11.039

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    13) Product Images from "Reconstituting ParA/ParB-mediated transport of DNA cargo"

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    Journal: Methods in cell biology

    doi: 10.1016/bs.mcb.2015.01.021

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.
    Figure Legend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Techniques Used: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

    14) Product Images from "BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction"

    Article Title: BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00366

    Schematic diagram of strand-specific library synthesis mechanism . mRNAs are fragmented by heat and magnesium (1) and primed for cDNA synthesis by an adapter-containing oligonucleotide (2,3) . Size selection and cleanup removes unincorperated oligonucleotides and small cDNA fragments (4) . Transient duplex breathing at the terminus of the RNA-cDNA hybrid (5) facilitates interaction with the single-stranded portion of the 5-prime capturing adapter (6) and E. coli DNA Polymerase I catalyses its incorporation into a complete library molecule (7) .
    Figure Legend Snippet: Schematic diagram of strand-specific library synthesis mechanism . mRNAs are fragmented by heat and magnesium (1) and primed for cDNA synthesis by an adapter-containing oligonucleotide (2,3) . Size selection and cleanup removes unincorperated oligonucleotides and small cDNA fragments (4) . Transient duplex breathing at the terminus of the RNA-cDNA hybrid (5) facilitates interaction with the single-stranded portion of the 5-prime capturing adapter (6) and E. coli DNA Polymerase I catalyses its incorporation into a complete library molecule (7) .

    Techniques Used: Selection

    Related Articles

    Incubation:

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood
    Article Snippet: .. After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added. ..

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. Modified plasmids were cut by incubation with Fast Digest SpeI and Kpn1 restriction enzymes (Fermentas) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-50 microcolumns (GE Healthcare) followed by incubation in 10% piperidine at 95°C for 20 min and repeated lyophilizations, samples were analyzed by electrophoresis on 12% denaturing polyacrylamide gels without fragment purification, allowing the shorter fragment to run off the gel.

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. For gel shift studies of plasmid fragments, after incubation with PNAs plasmids were cut by incubation with Fast Digest SpeI and PstI restriction enzymes (Fermentas, Hanover, MD, USA) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-25 microcolumns samples were run with gel electrophoresis.

    Article Title: Relationship between 3?-Azido-3?-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase
    Article Snippet: .. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (0.3 U; USB Corp.) and all four dNTPs (100 μM each). ..

    Labeling:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. Modified plasmids were cut by incubation with Fast Digest SpeI and Kpn1 restriction enzymes (Fermentas) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-50 microcolumns (GE Healthcare) followed by incubation in 10% piperidine at 95°C for 20 min and repeated lyophilizations, samples were analyzed by electrophoresis on 12% denaturing polyacrylamide gels without fragment purification, allowing the shorter fragment to run off the gel.

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. For gel shift studies of plasmid fragments, after incubation with PNAs plasmids were cut by incubation with Fast Digest SpeI and PstI restriction enzymes (Fermentas, Hanover, MD, USA) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-25 microcolumns samples were run with gel electrophoresis.

    Random Hexamer Labeling:

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells
    Article Snippet: .. 10 U of DNA Polymerase I (Invitrogen, Carlsbad, CA) was added to the denatured amplicons along with 250 nanograms of unmodified random hexamer primer, 1 mM dNTPs, 1x Ampligase buffer (Epicentre, Madison, Wi), and 1x NEB buffer 2 (NEB, Cambridge, MA). ..

    other:

    Article Title: A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA
    Article Snippet: DNA polymerase I (0.2 U/µl, 20 minutes, RT, Fermentas), a buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and alternatively 0.05 mM Alexa 555-dUTP, biotin-dUTP or digoxigenin-dUTP; when biotin-dUTP and digoxigenin-dUTP were used, we also added dTTP or 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP).

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ
    Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

    Electrophoretic Mobility Shift Assay:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. For gel shift studies of plasmid fragments, after incubation with PNAs plasmids were cut by incubation with Fast Digest SpeI and PstI restriction enzymes (Fermentas, Hanover, MD, USA) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-25 microcolumns samples were run with gel electrophoresis.

    Modification:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. Modified plasmids were cut by incubation with Fast Digest SpeI and Kpn1 restriction enzymes (Fermentas) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-50 microcolumns (GE Healthcare) followed by incubation in 10% piperidine at 95°C for 20 min and repeated lyophilizations, samples were analyzed by electrophoresis on 12% denaturing polyacrylamide gels without fragment purification, allowing the shorter fragment to run off the gel.

    Plasmid Preparation:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: .. For gel shift studies of plasmid fragments, after incubation with PNAs plasmids were cut by incubation with Fast Digest SpeI and PstI restriction enzymes (Fermentas, Hanover, MD, USA) for 20 min at 37°C followed by 3′-32 P labeling with [α-32 P]-dCTP (Perkin Elmer) by Klenow fragment of DNA polymerase I (Fermentas). .. After purification on G-25 microcolumns samples were run with gel electrophoresis.

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  • 99
    Thermo Fisher rnase r
    Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).
    Rnase R, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 445 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna polymerase i
    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS <t>DNA</t> synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sybr green i nucleic acid gel stain
    Analysis of cfDNA concentration in plasma of patients with renal carcinoma and controls. cfDNA concentrations were determined by measuring the fluorescence level of intercalated <t>SYBR</t> <t>Green</t> I dye (a) and by qPCR (c). ROC curve analysis of cfDNA concentration in cancer patients compared with the control group ((b) fluorescence test; (d) qPCR).
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    Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).

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

    Article Title: Circular DNA tumor viruses make circular RNAs

    doi: 10.1073/pnas.1811728115

    Figure Lengend Snippet: Subcellular localization of viral circRNAs. ( A ) Nuclear vs. cytoplasmic localization of viral circRNAs. RNA extracted from nuclear (Nuc) and cytoplasmic (Cyto) fractions of the KSHV and EBV coinfected BC1 cell line was either treated (+) or untreated (−) with RNase R. BSJs spanning PCR products from intron-retaining circBART_1.1 and circBART_2.1 were detected mainly in the nuclear fraction. Exonic circBART_1.2 and circBART_2.2 were found in both fractions. The circvIRF4 junction-spanning PCR products were detected in both fractions. Protein immunoblotting for lamin A/C (nuclear) and LAMP1 (cytoplasmic) was used to confirm fractionation quality. ( B ) Polysome profile for viral circRNAs. RNA polysome sucrose gradient profile (254-nm absorbance) of BC1 cell lysates after treatment with CHX ( Top ). Fraction 1 is at the top of the gradient (free mRNAs) and 12 corresponds to the bottom of the gradient. Polysome RNA fractions are represented by fractions 9 to 12. Quantitative PCR revealed that circvIRF4, circBART_1, and circBART_2 RNAs were not detected in polysomal fractions, whereas mRNAs for translated v-cyclin, LMP2, and GAPDH proteins preferentially fractionated with polysomes ( Bottom ).

    Article Snippet: One microgram of DNase digested RNA was either treated or untreated with Rnase R and reverse-transcribed using SuperScript IV (Thermo Fisher) with random hexamers in a total volume of 20 µL, according to the manufacturer’s protocol.

    Techniques: Polymerase Chain Reaction, Fractionation, Real-time Polymerase Chain Reaction

    Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Journal: Scientific Reports

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    doi: 10.1038/s41598-018-20421-1

    Figure Lengend Snippet: Genetic cargo of mRPC derived extracellular vesicles. ( A ) A 1.5% denaturing agarose gel loaded with total RNA from mRPCs and EVs. Total RNA from EVs consisted primarily of species below 800 nucleotides (nt) lacking 28S and 18S rRNA. EVs were treated with RNase and no difference was detected when compared with non-treated EVs, indicating the RNA of EVs was enclosed within the vesicle membrane. ( B ) Transcription factors, a cell-cycle regulator and intermediate filaments were identified in both mRPCs and EVs included Pax6, Hes1, Sox2, Ki67, GFAP and Nestin. The transcription factors identified are collectively involved in facilitating mRPC multipotency, cell-cycle and fate specification during retinogenesis. GFP, GAPDH and β-actin mRNAs were also detected in mRPCs and EVs. Next, the presence of miRNAs with established expression and function during retinogensis were chosen for analysis. ( C ) Selected miRNA species analyzed included Let7d, miR-9, miR-182 and miR-204. U6 snRNA was used as control. Data presented were combined from four independent replicates.

    Article Snippet: Prior to RNA and miRNA extraction, EVs were treated with 100 ug/ml RNAse (Thermoscientific) for 30 min at 37 °C according to manufacturer’s instruction.

    Techniques: Derivative Assay, Agarose Gel Electrophoresis, Expressing

    Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Journal: Scientific Reports

    Article Title: Retinal progenitor cells release extracellular vesicles containing developmental transcription factors, microRNA and membrane proteins

    doi: 10.1038/s41598-018-20421-1

    Figure Lengend Snippet: Extracellular vesicle internalization and transfer of GFP mRNA ( A ) Super resolution 3D reconstruction of GFP+ mRPC following 24 h incubation with PKH26 labeled extracellular vesicles. Red vesicles are visibly localized near the cell surface and within cytoplasm. In the XZ axis, GFP (green), EVs (red) and nuclei (blue, DAPI). ( B ) same as ( A ) with GFP (FITC) channel removed to increase visibility of PKH26 (TRITC) labeled EVs. Each panel contains three cross-sectional views (xy, xz, and yz). Scale: 5 µm. ( C ) RT-PCR analysis of GFP mRNA transfer between GFP+ mRPCs and non-GFP hRPCs. Non-GFP hRPCs served as negative control; GFP+ mRPCs served as postive control. GAPDH served as the internal control gene. EVs were treated using an RNase-Free DNase Set to remove DNA comtamination before cDNA synthesis. ( D ) Intensities of RT-PCR images were measured with ImageJ software and normalized to GAPDH. Relative levels of hRPC GFP after transfer of EVs is significantly higher than negative control.

    Article Snippet: Prior to RNA and miRNA extraction, EVs were treated with 100 ug/ml RNAse (Thermoscientific) for 30 min at 37 °C according to manufacturer’s instruction.

    Techniques: Incubation, Labeling, Reverse Transcription Polymerase Chain Reaction, Negative Control, Software

    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Journal: Journal of Virological Methods

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    doi: 10.1016/j.jviromet.2012.11.039

    Figure Lengend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Article Snippet: The efficiency of different SS synthesis enzymes, T7 Polymerase (Thermo Scientific, Vilnius, Lithuania), DNA Polymerase I (Thermo Scientific, Vilnius, Lithuania), and Sequenase 2.0 (Affymetrix, United Kingdom), was evaluated by means of densitometry following bands separation on a 1.5% agarose gel.

    Techniques: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    Analysis of cfDNA concentration in plasma of patients with renal carcinoma and controls. cfDNA concentrations were determined by measuring the fluorescence level of intercalated SYBR Green I dye (a) and by qPCR (c). ROC curve analysis of cfDNA concentration in cancer patients compared with the control group ((b) fluorescence test; (d) qPCR).

    Journal: Disease Markers

    Article Title: Concentration and Methylation of Cell-Free DNA from Blood Plasma as Diagnostic Markers of Renal Cancer

    doi: 10.1155/2016/3693096

    Figure Lengend Snippet: Analysis of cfDNA concentration in plasma of patients with renal carcinoma and controls. cfDNA concentrations were determined by measuring the fluorescence level of intercalated SYBR Green I dye (a) and by qPCR (c). ROC curve analysis of cfDNA concentration in cancer patients compared with the control group ((b) fluorescence test; (d) qPCR).

    Article Snippet: Specifically, 5 μ L of a sample or the same volume of a standard dilution of genomic DNA (Human HCT116 DKO Nonmethylated DNA) with known concentration (0 ng/mL and 9 serial dilutions from 1 to 256 ng/mL) was added to 195 μ L of a SYBR Green I solution (Cat. number S7585, Thermo Fisher Scientific, USA) in PBS buffer (1 : 10,000) and to black 96-well plates (PAA, Cat. number PAA30296X, Austria) and incubated for 10 min. Two to three identical mixtures were prepared from each sample or standard for greater accuracy.

    Techniques: Concentration Assay, Fluorescence, SYBR Green Assay, Real-time Polymerase Chain Reaction