dna polymerase i  (Thermo Fisher)


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  • 99
    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:
    EP0051
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis|DNA Labeling|Nucleic Acid Labeling & Oligo Synthesis|cDNA Libraries & Library Construction|Mutagenesis
    Size:
    300 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Polymerases
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher dna polymerase i
    Gel shift studies of plasmid <t>DNA</t> 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.
    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 99 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    dna polymerase i - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    Related Articles

    Clone Assay:

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    Article Snippet: Recombinant p53, Δ133p53, Δ40p53 and APE1 were generated by cloning cDNAs into a His-tagged expression vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: To obtain Ctrl construct, a PCR fragment was amplified with F15/R15 primers, digested with MluI and XhoI, and cloned into MluI and XhoI-digested pmirGLO. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Centrifugation:

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA). .. First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA).

    Amplification:

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
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    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
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    Article Title: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays
    Article Snippet: When starting with 20 ng total RNA or less, two rounds of amplification were performed. .. Briefly, 10 μl cDNA sample were mixed with 90 μl Klenow mixture to yield a reaction mixture that contained 1× random primer solution (Invitrogen), 200 μM each of dATP, dCTP and dGTP, 50 μM dTTP (Amersham Biosciences), 30 μM Cy3- or Cy5-dUTP (Amersham Biosciences) and 1.0 U/μl Klenow fragment (Invitrogen).

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: To obtain Ctrl construct, a PCR fragment was amplified with F15/R15 primers, digested with MluI and XhoI, and cloned into MluI and XhoI-digested pmirGLO. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Filtration:

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Reaction clean-up was carried out by gel filtration using Sephadex G50-columns (Mini Quick Spin DNA Columns; Roche #11814419001) according to the manufacturer’s protocol. .. Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL.

    Synthesized:

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: In brief, 1 μl oligo dT primer (100 μM) and 10 μl (10 μg) total RNAs were combined and denatured at 70°C for 5 min and renatured by cooling the mixture in ice. .. First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA). .. For the synthesis of Cy3-labeled target DNA fragments, 1 μg double-stranded cDNA was mixed with 30 μl (1 optical density) Cy3-9mer primer (Sigma-Aldrich, USA) and denatured by heating at 98°C for 10 min.

    Construct:

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: Paragraph title: Luciferase constructs ... The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Microarray:

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: Paragraph title: cDNA synthesis and microarray hybridization ... First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA).

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    Article Snippet: Paragraph title: 4.8. Microarray Analyses of Sorted CD4+ CD25+ T Cells ... Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Random Hexamer Labeling:

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells
    Article Snippet: 1.5 μL of NS buffer was added on ice to neutralize the solution. .. 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). .. The solution was incubated at 37 °C for 1 hour, allowing second strand synthesis.

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: First strand synthesis was performed using Random Hexamer Primers (Invitrogen, #48190-011) and SuperScript II (Invitrogen, #18064-014). .. Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen). .. Double-stranded cDNA was amplified using the Promega P1300 RiboMax Kit, for T7 amplification (Promega, Madison, WI, USA).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen). .. Double-stranded cDNA was amplified using the Promega P1300 RiboMax Kit, for T7 amplification (Promega, Madison, WI, USA).

    Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins
    Article Snippet: After first strand synthesis, the second strand was made using RNase H and DNA polymerase I (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ instructions. .. The resultant double-stranded cDNA was fragmented, ligated with Illumina sequencing adapters and sequenced in 36 cycles using the Genome Analyzer II platform at the UNC High Throughput Sequencing Facility.

    Luciferase:

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: Paragraph title: Luciferase constructs ... The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Infection:

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Splenocytes from both infected and control mice were isolated and pooled (n = 6 per group) on day 7 or 42, post infection. .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Splenocytes from both infected and control mice were isolated and pooled ( n = 6 per group) on day 7 or 42, post infection. .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Expressing:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Recombinant p53, Δ133p53, Δ40p53 and APE1 were generated by cloning cDNAs into a His-tagged expression vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Sequencing was performed on an Illumina NextSeq 500 by Stanford Functional Genomics Facility.

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen). .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen). .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: One ATG codon in e5΄UTR2 was mutated [G to T; chr3: 60501374 (mm10)] using the forward primer to prevent expression of a shorter protein that could delay the translation of luciferase. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Modification:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: Reactions were stopped with 5× stop solution on ice (1.5 M sodium acetate pH 5.2, 1 M β-mercaptoethanol, 100 μg/ml yeast t-RNA) followed by ethanol precipitation and washing with 70% ethanol. .. 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: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays
    Article Snippet: Thereafter, second-strand synthesis, cDNA purification, in vitro transcription, aRNA clean-up and third round reverse transcription (primed with random hexamers) were performed as described above. cDNA labelling by Klenow fragment was performed using the Bioprime Kit (Invitrogen), but with a modified protocol. .. Briefly, 10 μl cDNA sample were mixed with 90 μl Klenow mixture to yield a reaction mixture that contained 1× random primer solution (Invitrogen), 200 μM each of dATP, dCTP and dGTP, 50 μM dTTP (Amersham Biosciences), 30 μM Cy3- or Cy5-dUTP (Amersham Biosciences) and 1.0 U/μl Klenow fragment (Invitrogen).

    Hybridization:

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: Paragraph title: cDNA synthesis and microarray hybridization ... First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA).

    Transfection:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Paragraph title: Cell culture, d4T treatments, p53 RNAi transfection and reagents ... Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Ligation:

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: Δ1 and Δ2 were obtained by digestion of Ctrl with SmaI and XbaI. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation. .. Deleted sequences in Δ1 and Δ2 constructs are listed in .

    Cell Culture:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Paragraph title: Cell culture, d4T treatments, p53 RNAi transfection and reagents ... Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Generated:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Recombinant p53, Δ133p53, Δ40p53 and APE1 were generated by cloning cDNAs into a His-tagged expression vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    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: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation. .. Deleted sequences in Δ1 and Δ2 constructs are listed in .

    other:

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

    Sequencing:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Libraries were end-repaired, 3′ A-tailed, and ligated to NEBNext Multiplex Oligo Adaptors (NEB E7335S).

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: The sequence contained 65 nucleotides of e5΄UTR2 and 863 nucleotides of e1. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL. .. Reactions were incubated for 2 hr at 16°C then purified with by the MiniElute Reaction clean-up kit (Qiagen, #28004), eluted in 2 × 10 µL volumes in a single reaction tube.

    Recombinant:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Recombinant p53, Δ133p53, Δ40p53 and APE1 were generated by cloning cDNAs into a His-tagged expression vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Multiplex Assay:

    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. .. 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.

    Radioactivity:

    Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication
    Article Snippet: For dGTP and dCTP determination 1 U of Thermo Sequenase DNA Polymerase (GE Healthcare) was used; for dTTP determination 1.25 U Klenow Fragment (Thermo Fisher Scientific) and for dATP determination 0.625 U Klenow Fragment (Thermo Fisher Scientific) was used. .. For dGTP and dCTP determination 1 U of Thermo Sequenase DNA Polymerase (GE Healthcare) was used; for dTTP determination 1.25 U Klenow Fragment (Thermo Fisher Scientific) and for dATP determination 0.625 U Klenow Fragment (Thermo Fisher Scientific) was used.

    RNA Sequencing Assay:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Paragraph title: RNA Sequencing ... Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Paragraph title: cDNA library preparation for RNA-Seq ... Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL.

    Methylation:

    Article Title: Corrupted coordination of epigenetic modifications leads to diverging chromatin states and transcriptional heterogeneity in CLL
    Article Snippet: Digested DNA was filled-in and A-tailed at the 3′ sticky ends in 8.5 μL final volume of 1× CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas). .. Digested DNA was filled-in and A-tailed at the 3′ sticky ends in 8.5 μL final volume of 1× CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas).

    Isolation:

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: Thus, a total of 12 samples were subjected to total RNA isolation and used for microarray analyses. .. First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Total RNA of sorted CD4+ CD25+ cells was isolated using the Rneasy Mini Kit (Quiagen, Hilden, Germany), including the enzymatic digestion of DNA (Rnase-free Dnase Set, Quiagen), according to the manufacturer’s recommendations. .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Total RNA of sorted CD4+ CD25+ cells was isolated using the Rneasy Mini Kit (Quiagen, Hilden, Germany), including the enzymatic digestion of DNA (Rnase-free Dnase Set, Quiagen), according to the manufacturer’s recommendations. .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Flow Cytometry:

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: CD4+ CD25+ cells were flow cytometrically sorted on a BD FACSAria II instrument. .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: CD4+ CD25+ cells were flow cytometrically sorted on a BD FACSAria II instrument. .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Labeling:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: Reactions were stopped with 5× stop solution on ice (1.5 M sodium acetate pH 5.2, 1 M β-mercaptoethanol, 100 μg/ml yeast t-RNA) followed by ethanol precipitation and washing with 70% ethanol. .. 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: 5′-32 P labeled single stranded oligonucleotides bound with PNAs were run at room temperature in TBE buffer at 120 V. Gels were quantified using BAS-2500 Bioimager (FUJI Medical Systems USA, Stamford, CT, USA). .. 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.

    Purification:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Purified protein was quantified using silver-stained SDS-PAGE with bovine serum albumin standards. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Article Title: Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection
    Article Snippet: In brief, 1 μl oligo dT primer (100 μM) and 10 μl (10 μg) total RNAs were combined and denatured at 70°C for 5 min and renatured by cooling the mixture in ice. .. First-strand DNA was synthesized by adding 4 μl 5 X First Strand Buffer, 1 μl RiboLockTM Ribonuclease Inhibitor, 2 μl 10 mM dNTP mix, and 1 μl RevertAidTM H Minus M-MuLV Reverse Transcriptase enzyme and incubating at 42°C for 1 h. The reaction was stopped by heating at 70°C for 10 min. To synthesize the second strand, 66.7 μl nuclease free water, 5 μl 10 X reaction buffer for DNA Polymerase I (Fermentas, Lithuania), 5 μl 10 X T4 DNA ligase buffer (Takara, Japan), 3 μl 10 unit/ul DNA Polymerase I (Fermentas, Lithuania), 0.2 μl 5 unit/μl Ribonuclease H (Fermentas, Lithuania), and 0.1 μl 350 unit/μl T4 DNA ligase (Takara, Japan) were added to the first-strand reaction mixture and the reaction was performed at 15°C for 2 h. The double-stranded cDNA mixture was purified using the MinElute Reaction Cleanup Kit (QIAGEN, USA). .. For the synthesis of Cy3-labeled target DNA fragments, 1 μg double-stranded cDNA was mixed with 30 μl (1 optical density) Cy3-9mer primer (Sigma-Aldrich, USA) and denatured by heating at 98°C for 10 min.

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Polyadenylated RNA was selected using Ambion Dynabeads mRNA Purification Kit (Life Technologies 61006) and fragmented with Fragmentation Buffer (Ambion, #AM8740). .. Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014).

    Article Title: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays
    Article Snippet: Thereafter, second-strand synthesis, cDNA purification, in vitro transcription, aRNA clean-up and third round reverse transcription (primed with random hexamers) were performed as described above. cDNA labelling by Klenow fragment was performed using the Bioprime Kit (Invitrogen), but with a modified protocol. .. Briefly, 10 μl cDNA sample were mixed with 90 μl Klenow mixture to yield a reaction mixture that contained 1× random primer solution (Invitrogen), 200 μM each of dATP, dCTP and dGTP, 50 μM dTTP (Amersham Biosciences), 30 μM Cy3- or Cy5-dUTP (Amersham Biosciences) and 1.0 U/μl Klenow fragment (Invitrogen).

    Article Title: RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins
    Article Snippet: The IPs, RNA purification and reverse transcription were done essentially as described [ ]. .. After first strand synthesis, the second strand was made using RNase H and DNA polymerase I (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ instructions.

    Polymerase Chain Reaction:

    Article Title: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: To obtain Ctrl construct, a PCR fragment was amplified with F15/R15 primers, digested with MluI and XhoI, and cloned into MluI and XhoI-digested pmirGLO. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Electrophoretic Mobility Shift Assay:

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: 5′-32 P labeled single stranded oligonucleotides bound with PNAs were run at room temperature in TBE buffer at 120 V. Gels were quantified using BAS-2500 Bioimager (FUJI Medical Systems USA, Stamford, CT, USA). .. 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.

    cDNA Library Assay:

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Paragraph title: cDNA library preparation for RNA-Seq ... Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL.

    Mouse Assay:

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Splenocytes from both infected and control mice were isolated and pooled (n = 6 per group) on day 7 or 42, post infection. .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Splenocytes from both infected and control mice were isolated and pooled ( n = 6 per group) on day 7 or 42, post infection. .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    SDS Page:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Purified protein was quantified using silver-stained SDS-PAGE with bovine serum albumin standards. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Plasmid Preparation:

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T
    Article Snippet: Recombinant p53, Δ133p53, Δ40p53 and APE1 were generated by cloning cDNAs into a His-tagged expression vector (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. .. Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: Paragraph title: Chemical Probing of plasmid DNA ... 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).

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: 5′-32 P labeled single stranded oligonucleotides bound with PNAs were run at room temperature in TBE buffer at 120 V. Gels were quantified using BAS-2500 Bioimager (FUJI Medical Systems USA, Stamford, CT, USA). .. 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: Autoregulation of MBNL1 function by exon 1 exclusion from MBNL1 transcript
    Article Snippet: Obtained Mbnl1 fragment was digested with HindIII and cloned into HindIII digested pGL4.51 vector. .. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation.

    Software:

    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.

    Functional Assay:

    Article Title: Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma
    Article Snippet: Second strand synthesis was performed using DNA Pol I (Invitrogen #18010-025) and RNA was removed using RNaseH (Invitrogen #18021-014). .. Libraries were end-repaired, 3′ A-tailed, and ligated to NEBNext Multiplex Oligo Adaptors (NEB E7335S).

    In Vitro:

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen). .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen). .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Article Title: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays
    Article Snippet: Thereafter, second-strand synthesis, cDNA purification, in vitro transcription, aRNA clean-up and third round reverse transcription (primed with random hexamers) were performed as described above. cDNA labelling by Klenow fragment was performed using the Bioprime Kit (Invitrogen), but with a modified protocol. .. Briefly, 10 μl cDNA sample were mixed with 90 μl Klenow mixture to yield a reaction mixture that contained 1× random primer solution (Invitrogen), 200 μM each of dATP, dCTP and dGTP, 50 μM dTTP (Amersham Biosciences), 30 μM Cy3- or Cy5-dUTP (Amersham Biosciences) and 1.0 U/μl Klenow fragment (Invitrogen).

    Ethanol Precipitation:

    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). .. 1 U of Ampligase was added to seal nicks and the reaction was incubated first at 37 °C for 10 minutes and then at 65 °C for 10 minutes.

    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). .. 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).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: RNA was concentrated by ethanol precipitation following elution, and RNA integrity was tested using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). .. Second-strand cDNA synthesis was performed using DNA-polymerase I (Escherichia coli DNA ligase; Invitrogen).

    Article Title: Respiratory Bordetella bronchiseptica Carriage is Associated with Broad Phenotypic Alterations of Peripheral CD4+CD25+ T Cells and Differentially Affects Immune Responses to Secondary Non-Infectious and Infectious Stimuli in Mice
    Article Snippet: RNA was concentrated by ethanol precipitation following elution, and RNA integrity was tested using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). .. Second-strand cDNA synthesis was performed using DNA-polymerase I ( Escherichia coli DNA ligase; Invitrogen).

    Incubation:

    Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication
    Article Snippet: For dGTP and dCTP determination 1 U of Thermo Sequenase DNA Polymerase (GE Healthcare) was used; for dTTP determination 1.25 U Klenow Fragment (Thermo Fisher Scientific) and for dATP determination 0.625 U Klenow Fragment (Thermo Fisher Scientific) was used. .. Reactions were incubated for 1 hr either at 48°C (for dGTP and dCTP mixtures) or at room temperature (for dTTP and dATP mixtures).

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells
    Article Snippet: 1.5 μL of ALS buffer was added to the extracted amplicons to denature the DNA followed by a 3-minute incubation at room temperature. .. 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).

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood
    Article Snippet: An amount of 76 µl of dilution mix containing 70.5 mM Tris–HCl, 17.7 mM ammonium sulfate, 1.05 mM dithiothreitol and 40 µM of each dNTP's were added. .. 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. .. The extension reactions were run at 37°C for another 20 min followed by a 10-min heat-inactivation step at 80°C.

    Article Title: Quadruplex formation is necessary for stable PNA invasion into duplex DNA of BCL2 promoter region
    Article Snippet: Reactions were stopped with 5× stop solution on ice (1.5 M sodium acetate pH 5.2, 1 M β-mercaptoethanol, 100 μg/ml yeast t-RNA) followed by ethanol precipitation and washing with 70% ethanol. .. 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: 5′-32 P labeled single stranded oligonucleotides bound with PNAs were run at room temperature in TBE buffer at 120 V. Gels were quantified using BAS-2500 Bioimager (FUJI Medical Systems USA, Stamford, CT, USA). .. 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: Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays
    Article Snippet: First-strand cDNA was mixed with 100 ng (dT)-T7 primer in a final volume of 11 μl, incubated 10 min at 42°C and chilled on ice. .. Briefly, 10 μl cDNA sample were mixed with 90 μl Klenow mixture to yield a reaction mixture that contained 1× random primer solution (Invitrogen), 200 μM each of dATP, dCTP and dGTP, 50 μM dTTP (Amersham Biosciences), 30 μM Cy3- or Cy5-dUTP (Amersham Biosciences) and 1.0 U/μl Klenow fragment (Invitrogen).

    Article Title: Corrupted coordination of epigenetic modifications leads to diverging chromatin states and transcriptional heterogeneity in CLL
    Article Snippet: DNA was incubated with 10 units of the restriction enzyme Msp1 (Fermentas) in 6.5 μL final volume reaction during 90 min at 37 °C. .. Digested DNA was filled-in and A-tailed at the 3′ sticky ends in 8.5 μL final volume of 1× CutSmart with 2.5 units of Klenow fragment (Exo-, Fermentas).

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL. .. Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL.

    Evaporation:

    Article Title: Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication
    Article Snippet: Samples were then boiled, centrifuged and the supernatant was dried by centrifugal evaporation and finally dissolved in H2 O. .. For dGTP and dCTP determination 1 U of Thermo Sequenase DNA Polymerase (GE Healthcare) was used; for dTTP determination 1.25 U Klenow Fragment (Thermo Fisher Scientific) and for dATP determination 0.625 U Klenow Fragment (Thermo Fisher Scientific) was used.

    Concentration Assay:

    Article Title: Combined transcriptome and proteome profiling reveals specific molecular brain signatures for sex, maturation and circalunar clock phase
    Article Snippet: Fragment size distribution and concentration was analysed by Bioanalyzer (mRNA protocol). .. Second-strand cDNA was synthesised in a final reaction containing the first strand cDNA reaction and final concentrations of: 550 µM of each dATP, cGTP, dCTP (Thermo Scientific, #R0181), and dUTP (deoxy-UTP sodium salt; Roche, #11934554001); 0.300 U DNA Polymerase I (E.coli ; Invitrogen, #18010–025), 0.073 U E. coli DNA ligase (Invitrogen, #18052–019), and 0.015 U RNase H (Promega, #M4281); 20 µL 5X second strand buffer (Invitrogen, #10812–014), in a final volume of 100 µL.

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    Thermo Fisher klenow fragment
    Klenow Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/Thermo Fisher
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    Thermo Fisher genomic dna digestion mix
    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary <t>DNA</t> (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease <t>(DNase</t> I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Genomic Dna Digestion Mix, supplied by Thermo Fisher, 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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    Thermo Fisher antisense
    Gβ 1 , GAP43, and Gγ 13 gene transcript expression distribution in the MOE analysis using line intensity scan analysis. (A) Representative line intensity scans of RISH signals of Gβ 1 , GAP43, and Gγ 13 <t>antisense</t> probe labeling corresponding to line segments marked from “a” to “b” in insets. Insets: images of typical RISH labeling. The apical region is indicated as “a” and the basal lamina indicated as “b”. (B) Comparison of averaged line intensity scans for Gβ 1 (red), Gγ 13 (dark grey) and GAP43 (blue) ( n = 3 mice per label). Error bars represent SEM on each point of averaged line scan of Gβ 1 (red), Gγ 13 (grey) and GAP43 (blue), respectively. Light-grey shaded box between dashed vertical lines corresponds to the region expressing GAP43. Strong, overlapping labels of Gβ 1 and Gγ 13 are observed in the upper portion of the OSN layer, where there is no label of GAP43 gene transcript. (C) Magnified view of the shaded region in (B) . Gγ 13 signal drops shapely and disappears, whereas Gβ 1 signal, although is reduced, remains and significantly overlaps with GAP43 label. “∫Gβ 1 ” and “∫Gγ 13 ” represent area integrals under Gβ 1 (red line) and Gγ 13 (grey line). The difference between these two distributions (over which area integrals are calculated) is statistically significant (Kolmogorov-Smirnov test, p
    Antisense, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Gβ 1 , GAP43, and Gγ 13 gene transcript expression distribution in the MOE analysis using line intensity scan analysis. (A) Representative line intensity scans of RISH signals of Gβ 1 , GAP43, and Gγ 13 antisense probe labeling corresponding to line segments marked from “a” to “b” in insets. Insets: images of typical RISH labeling. The apical region is indicated as “a” and the basal lamina indicated as “b”. (B) Comparison of averaged line intensity scans for Gβ 1 (red), Gγ 13 (dark grey) and GAP43 (blue) ( n = 3 mice per label). Error bars represent SEM on each point of averaged line scan of Gβ 1 (red), Gγ 13 (grey) and GAP43 (blue), respectively. Light-grey shaded box between dashed vertical lines corresponds to the region expressing GAP43. Strong, overlapping labels of Gβ 1 and Gγ 13 are observed in the upper portion of the OSN layer, where there is no label of GAP43 gene transcript. (C) Magnified view of the shaded region in (B) . Gγ 13 signal drops shapely and disappears, whereas Gβ 1 signal, although is reduced, remains and significantly overlaps with GAP43 label. “∫Gβ 1 ” and “∫Gγ 13 ” represent area integrals under Gβ 1 (red line) and Gγ 13 (grey line). The difference between these two distributions (over which area integrals are calculated) is statistically significant (Kolmogorov-Smirnov test, p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: Gβ 1 , GAP43, and Gγ 13 gene transcript expression distribution in the MOE analysis using line intensity scan analysis. (A) Representative line intensity scans of RISH signals of Gβ 1 , GAP43, and Gγ 13 antisense probe labeling corresponding to line segments marked from “a” to “b” in insets. Insets: images of typical RISH labeling. The apical region is indicated as “a” and the basal lamina indicated as “b”. (B) Comparison of averaged line intensity scans for Gβ 1 (red), Gγ 13 (dark grey) and GAP43 (blue) ( n = 3 mice per label). Error bars represent SEM on each point of averaged line scan of Gβ 1 (red), Gγ 13 (grey) and GAP43 (blue), respectively. Light-grey shaded box between dashed vertical lines corresponds to the region expressing GAP43. Strong, overlapping labels of Gβ 1 and Gγ 13 are observed in the upper portion of the OSN layer, where there is no label of GAP43 gene transcript. (C) Magnified view of the shaded region in (B) . Gγ 13 signal drops shapely and disappears, whereas Gβ 1 signal, although is reduced, remains and significantly overlaps with GAP43 label. “∫Gβ 1 ” and “∫Gγ 13 ” represent area integrals under Gβ 1 (red line) and Gγ 13 (grey line). The difference between these two distributions (over which area integrals are calculated) is statistically significant (Kolmogorov-Smirnov test, p

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: Expressing, Labeling, Mouse Assay

    Post-natal expression patterns of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 gene transcripts in sensory epithelium of the VNO . RISH images of Gβ 1 , Gγ 2 , Gγ 3 , Gγ 8 , and Gγ 13 antisense probe labeling at post-natal stages, P0 (a, d, g, j, m, respectively), P7 (b, e, h, k, n, respectively) and P14 (c, f, i, l, o, respectively). The expression patterns are largely unchanged although the signals of Gβ 1 , Gγ 2 , Gγ 3 , and Gγ 13 are increased postnatally. Scale for all panels: 50 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: Post-natal expression patterns of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 gene transcripts in sensory epithelium of the VNO . RISH images of Gβ 1 , Gγ 2 , Gγ 3 , Gγ 8 , and Gγ 13 antisense probe labeling at post-natal stages, P0 (a, d, g, j, m, respectively), P7 (b, e, h, k, n, respectively) and P14 (c, f, i, l, o, respectively). The expression patterns are largely unchanged although the signals of Gβ 1 , Gγ 2 , Gγ 3 , and Gγ 13 are increased postnatally. Scale for all panels: 50 μm.

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: Expressing, Labeling

    Post-natal expression of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 gene transcripts in the MOE . RISH images of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 antisense probe labeling at post-natal stages, P0 (a, d, g, j, respectively), P7 (b, e, h, k, respectively) and P14 (c, f, i, l, respectively). Note transient expression of Gγ 2 at P7 and 14 and largely unchanged expression patterns of Gβ 1 , Gγ 8 , and Gγ 13 . Scale for all panels: 50 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: Post-natal expression of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 gene transcripts in the MOE . RISH images of Gβ 1 , Gγ 2 , Gγ 8 , and Gγ 13 antisense probe labeling at post-natal stages, P0 (a, d, g, j, respectively), P7 (b, e, h, k, respectively) and P14 (c, f, i, l, respectively). Note transient expression of Gγ 2 at P7 and 14 and largely unchanged expression patterns of Gβ 1 , Gγ 8 , and Gγ 13 . Scale for all panels: 50 μm.

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: Expressing, Labeling

    RNA in situ hybridization analysis of Gβ and Gγ subunit gene transcript expression in the MOE. (A) Gβ subunit gene transcripts expression in the MOE: (a) A low magnification image of a MOE coronal section showing Gβ 1 gene transcript expression throughout the MOE. as: antisense probe. (a′) Gβ 1 gene transcript expression is absent in the respiratory epithelium (arrowhead). (b) Gβ 1 sense probe shows no labeling. (c) Higher-magnification image of Gβ 1 gene transcript labeling in the MOE Sus: sustentacular/supporting cell layer, OSN: olfactory sensory neuron layer. BL: basal lamina—black dashed line. (d and d′) images of antisense and sense probe labeling of Gβ 2 respectively, showing weak expression in supporting cell layer. se: sense probe. (e and e′) images of antisense and sense probe labeling of Gβ 5 , showing absence of Gβ 5 gene transcript. (B) RISH analysis of Gγ subunit gene transcripts in the MOE: (a) A low magnification image of a MOE coronal section labeled with Gγ 13 antisense probe, showing Gγ 13 gene transcript expression throughout the MOE. (a′) Gγ 13 gene transcript expression is absent in the adjacent respiratory epithelium (arrowhead). (b) Gγ 13 sense probe labeling. No signal was observed. (c) A higher-magnification image of Gγ 13 gene transcript labeling in the MOE. (d and d′) images of antisense and sense probe labeling of Gγ 2 , showing Gγ 2 gene transcript expression in supporting cells. (e and e′) images of antisense and sense probe labeling of Gγ 12 . The Gγ 12 gene transcript expression in supporting cells is weak. (f and f′) images of antisense and sense probe labeling of Gγ 8 , showing Gγ 8 gene transcript expression in immature OSN region. (g and g′) images of antisense and sense probe labeling of Gγ 3 . (h and h′) images of antisense and sense probe labeling of Gγ 5 . Note there is no labeling for both Gγ 3 and Gγ 5 gene transcripts in the MOE. In (A) and (B) , scale for (a, b) is 0.2 mm, (a′) is 50 μm, (c, d, d′, e, e′) is 20 μm; In (B), scale for (f, f′, g, g′, h, h′) is 20 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: RNA in situ hybridization analysis of Gβ and Gγ subunit gene transcript expression in the MOE. (A) Gβ subunit gene transcripts expression in the MOE: (a) A low magnification image of a MOE coronal section showing Gβ 1 gene transcript expression throughout the MOE. as: antisense probe. (a′) Gβ 1 gene transcript expression is absent in the respiratory epithelium (arrowhead). (b) Gβ 1 sense probe shows no labeling. (c) Higher-magnification image of Gβ 1 gene transcript labeling in the MOE Sus: sustentacular/supporting cell layer, OSN: olfactory sensory neuron layer. BL: basal lamina—black dashed line. (d and d′) images of antisense and sense probe labeling of Gβ 2 respectively, showing weak expression in supporting cell layer. se: sense probe. (e and e′) images of antisense and sense probe labeling of Gβ 5 , showing absence of Gβ 5 gene transcript. (B) RISH analysis of Gγ subunit gene transcripts in the MOE: (a) A low magnification image of a MOE coronal section labeled with Gγ 13 antisense probe, showing Gγ 13 gene transcript expression throughout the MOE. (a′) Gγ 13 gene transcript expression is absent in the adjacent respiratory epithelium (arrowhead). (b) Gγ 13 sense probe labeling. No signal was observed. (c) A higher-magnification image of Gγ 13 gene transcript labeling in the MOE. (d and d′) images of antisense and sense probe labeling of Gγ 2 , showing Gγ 2 gene transcript expression in supporting cells. (e and e′) images of antisense and sense probe labeling of Gγ 12 . The Gγ 12 gene transcript expression in supporting cells is weak. (f and f′) images of antisense and sense probe labeling of Gγ 8 , showing Gγ 8 gene transcript expression in immature OSN region. (g and g′) images of antisense and sense probe labeling of Gγ 3 . (h and h′) images of antisense and sense probe labeling of Gγ 5 . Note there is no labeling for both Gγ 3 and Gγ 5 gene transcripts in the MOE. In (A) and (B) , scale for (a, b) is 0.2 mm, (a′) is 50 μm, (c, d, d′, e, e′) is 20 μm; In (B), scale for (f, f′, g, g′, h, h′) is 20 μm.

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: RNA In Situ Hybridization, Expressing, Labeling

    RNA in situ hybridization analysis of Gβ and Gγ subunit gene transcript expression in the adult VNO. Top: RISH images of coronal sections of VNO labeled with antisense probes against Gβ 1 (a), Gγ 2 (b), Gγ 3 (c), Gγ 8 (d) and Gγ 13 (e); Bottom: Higher-magnification views of RISH images correlated to images in the top panels. Black dotted line represent basal lamina. Note that Gβ 1 and Gγ 8 are expressed throughout VSN layers, whereas Gγ 2 , Gγ 3 and Gγ 13 are expressed largely in the apical VSN zone. Scale: all top panels: 100 μm bottom panels: 50 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: RNA in situ hybridization analysis of Gβ and Gγ subunit gene transcript expression in the adult VNO. Top: RISH images of coronal sections of VNO labeled with antisense probes against Gβ 1 (a), Gγ 2 (b), Gγ 3 (c), Gγ 8 (d) and Gγ 13 (e); Bottom: Higher-magnification views of RISH images correlated to images in the top panels. Black dotted line represent basal lamina. Note that Gβ 1 and Gγ 8 are expressed throughout VSN layers, whereas Gγ 2 , Gγ 3 and Gγ 13 are expressed largely in the apical VSN zone. Scale: all top panels: 100 μm bottom panels: 50 μm.

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: RNA In Situ Hybridization, Expressing, Labeling

    Cell type-specific expression of Gβγ subunit gene transcripts in the VNO. (A) Confocal images of VNO coronal sections labeled with Gβ 1 (a, red) and Gα i2 (b, green) fluorescent probes in double in situ hybridization, respectively, and the overlay image (c). Gβ 1 is expressed in both apical, Gα i2 -expressing zone and basal zone lacking Gα i2 . (B) Confocal images of VNO coronal sections double-labeled with antisense probes against Gγ 2 , Gγ 3 , Gγ 13 (a, d, g, respectively, red) and Gα i2 (b, e, h, respectively, green) in in situ hybridization. The corresponding overlaid images are shown in c, f, i. Note that Gγ 2 , Gγ 3 , and Gγ 13 are colocalized in the apical, Gα i2 -expressing zone. Scale for all panels: 50 μm.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Expression profile of G-protein ?? subunit gene transcripts in the mouse olfactory sensory epithelia

    doi: 10.3389/fncel.2013.00084

    Figure Lengend Snippet: Cell type-specific expression of Gβγ subunit gene transcripts in the VNO. (A) Confocal images of VNO coronal sections labeled with Gβ 1 (a, red) and Gα i2 (b, green) fluorescent probes in double in situ hybridization, respectively, and the overlay image (c). Gβ 1 is expressed in both apical, Gα i2 -expressing zone and basal zone lacking Gα i2 . (B) Confocal images of VNO coronal sections double-labeled with antisense probes against Gγ 2 , Gγ 3 , Gγ 13 (a, d, g, respectively, red) and Gα i2 (b, e, h, respectively, green) in in situ hybridization. The corresponding overlaid images are shown in c, f, i. Note that Gγ 2 , Gγ 3 , and Gγ 13 are colocalized in the apical, Gα i2 -expressing zone. Scale for all panels: 50 μm.

    Article Snippet: The linearized plasmids were then used as templates in an in vitro transcription reaction, containing either digoxygenin-UTP (DIG) or fluorescein-UTP (FLU) to generate required antisense (+/+; Sph I; Sp6 RNA polymerase [Thermo Scientific]) and sense riboprobes (+/+; Nde I; T7 RNA polymerase [Thermo Scientific]).

    Techniques: Expressing, Labeling, In Situ, Hybridization