exonuclease i  (Thermo Fisher)


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  • 99
    Name:
    Exonuclease I
    Description:
    Thermo Scientific Exonuclease I (ExoI) degrades single-stranded DNA in a 3'→5' direction. It releases deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaves 5'-terminal dinucleotides intact.It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups.Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures:• prior to PCR product sequencing• for one-tube "megaprimer" PCR mutagenesis• Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminusNoteThe enzyme is not suitable for removing 3'-overhangs of dsDNA.
    Catalog Number:
    EN0581
    Price:
    None
    Applications:
    Cloning|PCR Cloning|Mutagenesis
    Size:
    4 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher exonuclease i
    Mutations in  STN1  result in abnormal telomere phenotypes.  (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P
    Thermo Scientific Exonuclease I (ExoI) degrades single-stranded DNA in a 3'→5' direction. It releases deoxyribonucleoside 5'-monophosphates in a stepwise manner and leaves 5'-terminal dinucleotides intact.It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups.Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures:• prior to PCR product sequencing• for one-tube "megaprimer" PCR mutagenesis• Removal of single-stranded DNA containing a 3'-hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single-stranded DNA with a 3'-hydroxyl terminusNoteThe enzyme is not suitable for removing 3'-overhangs of dsDNA.
    https://www.bioz.com/result/exonuclease i/product/Thermo Fisher
    Average 99 stars, based on 261 article reviews
    Price from $9.99 to $1999.99
    exonuclease i - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects"

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151618

    Mutations in  STN1  result in abnormal telomere phenotypes.  (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P
    Figure Legend Snippet: Mutations in STN1 result in abnormal telomere phenotypes. (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P

    Techniques Used: Hybridization, Fluorescence In Situ Hybridization

    2) Product Images from "Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination"

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv172

    The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.
    Figure Legend Snippet: The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.

    Techniques Used: Binding Assay, Incubation, Electrophoresis

    3) Product Images from "MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy"

    Article Title: MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061384

    Optimized RACE-PCR-based approach for TCR sequencing from single cells. (A) Sketch of the basic principle of the novel single-TCR sequencing strategy. Reverse transcription and exonuclease digest were consecutively done on-slide. Complete reactions were transferred to 96 well plates for tailing, anchor PCR and nested PCR round I and II. Alpha and beta chains were pre-amplified together during anchor PCR and in separate reactions for nested PCRs I and II. To change the reaction conditions for the first four steps of the protocol, the volume was increased to create optimal conditions for the subsequent enzyme. For nested PCR round I and II 1 µl of the previous reation volume was transferred to the next step. (B) Different priming strategies (standard oligo-dT/random hexamers, one gene-specific primer and three gene-specific primers per TCR chain) were tested with and without extra exonuclease-I digest of residual RT-primers. Decreasing numbers of human T cells, from 1000 to 1 as indicated served as template. (C)The anchor PCR step is used to prolong the oligo-dG overhang from the tailing step. PCR was tested without reverse primer in “linear” mode and with reverse primer in “exponential” mode of amplification. (D) Temperature switch during reverse transcription was tested. RT at a constant temperature of 51°C (upper row) was compared with a temperature increase form 51°C for 30 min to 70°C for 20 min (bottom row). (E) In seven independent single-cell PCR experiments a total number of 266 samples were processed. Numbers of samples yielding α- and/or β- chain products above the evaluated full-length cut-off size were calculated as a percentage of total samples per experiment. Mean values for all experiments taken together are indicated by horizontal lines.
    Figure Legend Snippet: Optimized RACE-PCR-based approach for TCR sequencing from single cells. (A) Sketch of the basic principle of the novel single-TCR sequencing strategy. Reverse transcription and exonuclease digest were consecutively done on-slide. Complete reactions were transferred to 96 well plates for tailing, anchor PCR and nested PCR round I and II. Alpha and beta chains were pre-amplified together during anchor PCR and in separate reactions for nested PCRs I and II. To change the reaction conditions for the first four steps of the protocol, the volume was increased to create optimal conditions for the subsequent enzyme. For nested PCR round I and II 1 µl of the previous reation volume was transferred to the next step. (B) Different priming strategies (standard oligo-dT/random hexamers, one gene-specific primer and three gene-specific primers per TCR chain) were tested with and without extra exonuclease-I digest of residual RT-primers. Decreasing numbers of human T cells, from 1000 to 1 as indicated served as template. (C)The anchor PCR step is used to prolong the oligo-dG overhang from the tailing step. PCR was tested without reverse primer in “linear” mode and with reverse primer in “exponential” mode of amplification. (D) Temperature switch during reverse transcription was tested. RT at a constant temperature of 51°C (upper row) was compared with a temperature increase form 51°C for 30 min to 70°C for 20 min (bottom row). (E) In seven independent single-cell PCR experiments a total number of 266 samples were processed. Numbers of samples yielding α- and/or β- chain products above the evaluated full-length cut-off size were calculated as a percentage of total samples per experiment. Mean values for all experiments taken together are indicated by horizontal lines.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Nested PCR, Amplification

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    Amplification:

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    DNA Synthesis:

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    Polymerase Chain Reaction:

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    Article Title: Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq
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    Electrophoresis:

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination
    Article Snippet: In a total of 10 μl reaction volume, 60-mer ssDNA (24 μM) was mixed with Dmc1 (12 μM) in a buffer B (50 mM Tris-HCl pH 7.5, 70 mM NaCl, 2 mM MgCl2 , 2 mM ATP) and incubated for 10 min at 37 o C. The Hop2–Mnd1 complex (2.5 μM) was added and incubated for 10 min followed by the addition of 10 units of exonuclease I (Thermo Scientific, 20 units/μl). .. After 20 min, the reaction was stopped by adding proteinase K (1 mg/ml) and further incubated for 20 min.

    Article Title: Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia
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    Article Title: Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools
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    Incubation:

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination
    Article Snippet: Protection assays with E. coli exonuclease I was performed as reported ( , ). .. In a total of 10 μl reaction volume, 60-mer ssDNA (24 μM) was mixed with Dmc1 (12 μM) in a buffer B (50 mM Tris-HCl pH 7.5, 70 mM NaCl, 2 mM MgCl2 , 2 mM ATP) and incubated for 10 min at 37 o C. The Hop2–Mnd1 complex (2.5 μM) was added and incubated for 10 min followed by the addition of 10 units of exonuclease I (Thermo Scientific, 20 units/μl). .. After 20 min, the reaction was stopped by adding proteinase K (1 mg/ml) and further incubated for 20 min.

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage
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    Article Title: Reprogramming Transcription via Distinct Classes of Enhancers Functionally Defined by eRNA
    Article Snippet: Then, the tube was added 0.5μl SUPERase-In, 3μl 0.1M DTT, 2μl 25mM MgCl2, 2μl 10× reverse transcription buffer, and 1μl superscript III reverse transcriptase (Invitrogen). .. After that, 4μl of Exonuclease I (Fermentas) was added into the reaction and the tube was incubated for 1 hr at 37°C to eliminate extra oNTI223. .. Then, RNA was eliminated by adding 1.8μl 1M NaOH and incubated for 20 min at 98°C.

    Article Title: Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq
    Article Snippet: Before library preparation, proteins were digested by incubation at 50 °C for 10 min. Proteinase K was then heat inactivated for 10 min at 80 °C. .. Purified cDNA was eluted in 17 µl and residual primers digested with Exonuclease I (Thermo Fisher) for 20 min at 37 °C.

    Expressing:

    Article Title: miR-152 Attenuates the Severity of Lupus Nephritis Through the Downregulation of Macrophage Migration Inhibitory Factor (MIF)-Induced Expression of COL1A1
    Article Snippet: Strand specific RT-PCR was used to detect the expression of MIF mRNA because of the presence of MIF-AS-1 on the complementary strand of MIF gene. .. Exonuclease-I (Thermo Fisher Scientific, USA) was then added to the system and the system was kept at 37°C for 30 min to excise the primers of cDNA.

    Acrylamide Gel Assay:

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination
    Article Snippet: In a total of 10 μl reaction volume, 60-mer ssDNA (24 μM) was mixed with Dmc1 (12 μM) in a buffer B (50 mM Tris-HCl pH 7.5, 70 mM NaCl, 2 mM MgCl2 , 2 mM ATP) and incubated for 10 min at 37 o C. The Hop2–Mnd1 complex (2.5 μM) was added and incubated for 10 min followed by the addition of 10 units of exonuclease I (Thermo Scientific, 20 units/μl). .. After 20 min, the reaction was stopped by adding proteinase K (1 mg/ml) and further incubated for 20 min.

    Hybridization:

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects
    Article Snippet: Paragraph title: In-gel hybridization analyses ... Exonuclease I treatment was performed overnight at 37°C with 200 U exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation.

    Concentration Assay:

    Article Title: MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy
    Article Snippet: Reverse transcription was performed for 20 min at 51°C followed by 30 min at 70°C on an AmpliSpeed PCR cycler. .. For primer exonuclease I digest, reaction volume was filled up to 1 µl resulting in a final concentration of 67 mM glycine-KOH (pH 9.5), 6.7 mM MgCl2 , 10 mM DTT and 1 U/ µl Exonuclease I (Fermentas). .. Exonuclease-I digest was performed for 30 min at 37°C and enzyme was inactivated for 20 min at 70°C on an AmpliSpeed PCR cycler.

    Infection:

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0.

    Digital PCR:

    Article Title: Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias
    Article Snippet: We performed CORD assay consisting of two-step treatments of DNA with multiple methylation-sensitive restriction enzymes followed by multiplex digital PCR [ ]. .. In the first step of enzyme treatment, 10 μL of eluted DNA (an amount of DNA equivalent to that in 80 μL serum) was digested for 16 hours at 37°C by the addition of 1 μL of GeneAmp 10x PCR Buffer II, 1 μL of 25 mmol/l MgCl2 , 10 units of Hha I, 10 units of Hpa II, and 20 units of exonuclease I (Exo I) (all from Thermo Fisher Scientific).

    DNA Sequencing:

    Article Title: The common truncation variant in pancreatic lipase related protein 2 (PNLIPRP2) is expressed poorly and does not alter risk for chronic pancreatitis
    Article Snippet: Paragraph title: DNA sequencing ... PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer.

    Sequencing:

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription
    Article Snippet: Paragraph title: Global Run-On Sequencing ... Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas).

    Article Title: Reprogramming Transcription via Distinct Classes of Enhancers Functionally Defined by eRNA
    Article Snippet: Paragraph title: Global run-on sequencing analysis (GRO-seq) ... After that, 4μl of Exonuclease I (Fermentas) was added into the reaction and the tube was incubated for 1 hr at 37°C to eliminate extra oNTI223.

    Article Title: Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools
    Article Snippet: Those specimens that did not amplify with ITS2 primers, and specimens corresponding to every novel ITS2 sequence were amplified with COI primers. .. The PCR product was purified using an enzyme cleanup: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Behaviour and molecular identification of Anopheles malaria vectors in Jayapura district, Papua province, Indonesia
    Article Snippet: The amplified fragments were purified using an enzyme clean-up: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. .. The amplified fragments were purified using an enzyme clean-up: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0.

    Article Title: Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir
    Article Snippet: Paragraph title: Single-genome sequencing (SGS) of p6-PR-RT from p24-positive VOA wells and from HIV DNA and cell-associated RNA in uncultured blood mononuclear cells ... The PCR product was treated with 20 U of Exonuclease I (Affymetrix) and 4 U of Shrimp Alkaline Phosphatase (Affymetrix).

    Article Title: The common truncation variant in pancreatic lipase related protein 2 (PNLIPRP2) is expressed poorly and does not alter risk for chronic pancreatitis
    Article Snippet: Cycling conditions were as follows: 15-min initial heat activation at 95 o C; 40 cycles of 30 s denaturation at 94 o C, 30 s annealing at 60 o C, and 60 s extension at 72 o C; and final extension for 5 min at 72 o C. Products were verified by 1.5% agarose gel electrophoresis. .. PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer. .. Amplicons containing the heterozygous c.1070-379delG variant were also sequenced with the reverse primer.

    Article Title: Neotropical bats that co-habit with humans function as dead-end hosts for dengue virus
    Article Snippet: PCR products were purified using Exonuclease I and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc.) following the manufacturer’s protocol. .. PCR products were purified using Exonuclease I and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc.) following the manufacturer’s protocol.

    Sonication:

    Article Title: Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir
    Article Snippet: The PCR product was treated with 20 U of Exonuclease I (Affymetrix) and 4 U of Shrimp Alkaline Phosphatase (Affymetrix). .. The PCR product was treated with 20 U of Exonuclease I (Affymetrix) and 4 U of Shrimp Alkaline Phosphatase (Affymetrix).

    DNA Extraction:

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects
    Article Snippet: Genomic DNA was extracted from peripheral blood samples using the G-DEX IIb genomic DNA extraction kit (iNtRon). .. Exonuclease I treatment was performed overnight at 37°C with 200 U exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation.

    Article Title: Behaviour and molecular identification of Anopheles malaria vectors in Jayapura district, Papua province, Indonesia
    Article Snippet: Genomic DNA was isolated from individual specimens using a CTAB DNA extraction. .. The amplified fragments were purified using an enzyme clean-up: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: Genomic DNA was isolated from individual mosquito heads and thoraces using a CTAB-based DNA extraction method. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0.

    Nucleic Acid Electrophoresis:

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription
    Article Snippet: Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas). .. The ssDNA template was amplified to generate DNA for sequencing by Phusion Hig-Fidelity DNA Polymerase (Thermo Science) with primers oNTI200 (5′-CAAGCAGAAGACGGCATA-3′) and oNTI201 (5′-AATG ATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3′).

    RNA Sequencing Assay:

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription
    Article Snippet: Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas). .. PCR product was purified and size selected (140-225 bp) by gel electrophoresis on a non-denaturing 8% polyacrylamide TBE gel (Invitrogen).

    Methylation:

    Article Title: Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias
    Article Snippet: We performed CORD assay consisting of two-step treatments of DNA with multiple methylation-sensitive restriction enzymes followed by multiplex digital PCR [ ]. .. In the first step of enzyme treatment, 10 μL of eluted DNA (an amount of DNA equivalent to that in 80 μL serum) was digested for 16 hours at 37°C by the addition of 1 μL of GeneAmp 10x PCR Buffer II, 1 μL of 25 mmol/l MgCl2 , 10 units of Hha I, 10 units of Hpa II, and 20 units of exonuclease I (Exo I) (all from Thermo Fisher Scientific).

    Isolation:

    Article Title: Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia
    Article Snippet: The ribosomal DNA internal transcribed spacer region two (rDNA ITS2) region was isolated with PCR using primers developed for differentiating other Anopheles species complexes [ ]. .. An enzyme cleanup was used to purify each PCR product: 2 U of Exonuclease 1 (USB Corporation, Cleveland, OH, USA), 1 U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: RB116: An RB1+ retinoblastoma cell line expressing primitive markers
    Article Snippet: Genomic DNA from RB116 cells was isolated using a Qiagen DNA mini kit (# 51,104). .. PCR reactions were cleaned by up SAP/Exo1 digestion: 1 unit of shrimp alkaline phosphatase (USB cat. # 70092Y) and 1 unit of exonuclease 1 (USB cat. # 70073Z; USB Corporation, Cleveland, OH) in 4 μl was added to each 10 μl PCR reaction.

    Article Title: Behaviour and molecular identification of Anopheles malaria vectors in Jayapura district, Papua province, Indonesia
    Article Snippet: This region of rDNA was isolated using PCR with ITS2A and ITS2B primers [ ]. .. The amplified fragments were purified using an enzyme clean-up: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: Genomic DNA was isolated from individual mosquito heads and thoraces using a CTAB-based DNA extraction method. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0.

    RNA Extraction:

    Article Title: Neotropical bats that co-habit with humans function as dead-end hosts for dengue virus
    Article Snippet: Viral RNA extraction, cDNA retro-transcription and DENV PCR were performed as described above. .. PCR products were purified using Exonuclease I and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc.) following the manufacturer’s protocol.

    Labeling:

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription
    Article Snippet: BrU labeled nascent transcripts were immunoprecipiated with anti-BrU agarose beads (Santa Cruz Biotech), washed, eluted and precipitated in ethanol. .. Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas).

    Purification:

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription
    Article Snippet: Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas). .. Subsequently, excess oNTI223 primers were removed by Exonuclease I (Fermentas).

    Article Title: Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools
    Article Snippet: The amplified fragments were visualized by electrophoresis on a 1% agarose gel. .. The PCR product was purified using an enzyme cleanup: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. .. The PCR products were sequenced directly (with one of the PCR primers) using Sanger sequencing on ABI 3730xl DNA Analyzer platform (PE Applied Biosystems, Warrington, England).

    Article Title: Behaviour and molecular identification of Anopheles malaria vectors in Jayapura district, Papua province, Indonesia
    Article Snippet: This region of rDNA was isolated using PCR with ITS2A and ITS2B primers [ ]. .. The amplified fragments were purified using an enzyme clean-up: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. .. PCR products were sequenced directly using Sanger sequencing on ABI 3730 xl DNA Analyzer platform (Applied Biosystems).

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: The ribosomal DNA internal transcribed spacer region (rDNA ITS2) was PCR-amplified using ITS2A and ITS2B primers [ ] that were developed to differentiate Anopheles cryptic species, and then sequenced. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0. .. This cleanup mixture was incubated at 37 °C for 15 min, and then at 80 °C for 15 min to inactivate the enzymes.

    Article Title: Neotropical bats that co-habit with humans function as dead-end hosts for dengue virus
    Article Snippet: Gene segments were amplified with two sets of primers that amplify overlapping regions of mitochondrial cytochrome oxidase subunit I (COI), COI_short and COI_long, and one primer set for cytochrome b (Cyt b ) as described in Townzen et al. [ ]. .. PCR products were purified using Exonuclease I and Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc.) following the manufacturer’s protocol. .. Both strands of the amplicons were sequenced by Macrogen Inc. (Seoul, South Korea).

    Article Title: Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq
    Article Snippet: Barcoded cDNA was then pooled in 2 ml DNA LoBind tubes (Eppendorf) and cleaned up using SPRI beads. .. Purified cDNA was eluted in 17 µl and residual primers digested with Exonuclease I (Thermo Fisher) for 20 min at 37 °C. .. After heat inactivation for 10 min at 80 °C, 30 µl PCR master mix consisting of 1.25 U Terra direct polymerase (Clontech) 1.66 × Terra direct buffer and 0.33 µM SINGV6 primer (IDT) was added.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: miR-152 Attenuates the Severity of Lupus Nephritis Through the Downregulation of Macrophage Migration Inhibitory Factor (MIF)-Induced Expression of COL1A1
    Article Snippet: Strand specific RT-PCR was used to detect the expression of MIF mRNA because of the presence of MIF-AS-1 on the complementary strand of MIF gene. .. Exonuclease-I (Thermo Fisher Scientific, USA) was then added to the system and the system was kept at 37°C for 30 min to excise the primers of cDNA.

    Staining:

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination
    Article Snippet: In a total of 10 μl reaction volume, 60-mer ssDNA (24 μM) was mixed with Dmc1 (12 μM) in a buffer B (50 mM Tris-HCl pH 7.5, 70 mM NaCl, 2 mM MgCl2 , 2 mM ATP) and incubated for 10 min at 37 o C. The Hop2–Mnd1 complex (2.5 μM) was added and incubated for 10 min followed by the addition of 10 units of exonuclease I (Thermo Scientific, 20 units/μl). .. After 20 min, the reaction was stopped by adding proteinase K (1 mg/ml) and further incubated for 20 min.

    Nested PCR:

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0.

    Article Title: Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir
    Article Snippet: The nested PCR amplicon was ~1.5 kb spanning p6-PR-RT. .. The PCR product was treated with 20 U of Exonuclease I (Affymetrix) and 4 U of Shrimp Alkaline Phosphatase (Affymetrix).

    Real-time Polymerase Chain Reaction:

    Article Title: miR-152 Attenuates the Severity of Lupus Nephritis Through the Downregulation of Macrophage Migration Inhibitory Factor (MIF)-Induced Expression of COL1A1
    Article Snippet: Paragraph title: Real-Time PCR ... Exonuclease-I (Thermo Fisher Scientific, USA) was then added to the system and the system was kept at 37°C for 30 min to excise the primers of cDNA.

    Multiplex Assay:

    Article Title: Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias
    Article Snippet: We performed CORD assay consisting of two-step treatments of DNA with multiple methylation-sensitive restriction enzymes followed by multiplex digital PCR [ ]. .. In the first step of enzyme treatment, 10 μL of eluted DNA (an amount of DNA equivalent to that in 80 μL serum) was digested for 16 hours at 37°C by the addition of 1 μL of GeneAmp 10x PCR Buffer II, 1 μL of 25 mmol/l MgCl2 , 10 units of Hha I, 10 units of Hpa II, and 20 units of exonuclease I (Exo I) (all from Thermo Fisher Scientific).

    Agarose Gel Electrophoresis:

    Article Title: Host attraction and biting behaviour of Anopheles mosquitoes in South Halmahera, Indonesia
    Article Snippet: An enzyme cleanup was used to purify each PCR product: 2 U of Exonuclease 1 (USB Corporation, Cleveland, OH, USA), 1 U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. .. An enzyme cleanup was used to purify each PCR product: 2 U of Exonuclease 1 (USB Corporation, Cleveland, OH, USA), 1 U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools
    Article Snippet: The PCR product was purified using an enzyme cleanup: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. .. The PCR product was purified using an enzyme cleanup: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product.

    Article Title: Cow-baited tents are highly effective in sampling diverse Anopheles malaria vectors in Cambodia
    Article Snippet: The ribosomal DNA internal transcribed spacer region (rDNA ITS2) was PCR-amplified using ITS2A and ITS2B primers [ ] that were developed to differentiate Anopheles cryptic species, and then sequenced. .. PCR products were visualized on a 1 % agarose gel and purified by mixing 8 μl of PCR product with 2U of exonuclease 1 (USB Corporation, Cleveland, OH), 1U of shrimp alkaline phosphatase (USB Corporation), and 1.8 μl of ddH2 0. .. This cleanup mixture was incubated at 37 °C for 15 min, and then at 80 °C for 15 min to inactivate the enzymes.

    Article Title: The common truncation variant in pancreatic lipase related protein 2 (PNLIPRP2) is expressed poorly and does not alter risk for chronic pancreatitis
    Article Snippet: PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer. .. PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer.

    Ethanol Precipitation:

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects
    Article Snippet: Duplicated samples were electrophoresed in the same gel (0.7% agarose) and, after gel drying, hybridized separately with a C-rich [(TAACCC)3 ] and G-rich [(AGGGTT)3 ] probes. .. Exonuclease I treatment was performed overnight at 37°C with 200 U exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation. .. The hybridization signal intensity for each lane was determined before and after denaturation using Image J (National Institutes of Health).

    Activation Assay:

    Article Title: The common truncation variant in pancreatic lipase related protein 2 (PNLIPRP2) is expressed poorly and does not alter risk for chronic pancreatitis
    Article Snippet: PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer. .. PCR amplicons (5 μL) were treated with 1 μL FastAP Thermosensitive Alkaline Phosphatase and 0.5 μL Exonuclease I (Thermo Fisher Scientific, Waltham, MA) for 15 min at 37 o C and the reaction was stopped by heating the samples to 85 o C for 15 min. Sanger sequencing was performed using the forward PCR primers as sequencing primer.

    Lysis:

    Article Title: Sensitive and powerful single-cell RNA sequencing using mcSCRB-seq
    Article Snippet: Lysis buffer consisted of a 1:500 dilution of Phusion HF buffer (New England Biolabs), 1.25 µg/µl Proteinase K (Clontech), and 0.4 µM barcoded oligo-dT primer (E3V6NEXT, IDT). .. Purified cDNA was eluted in 17 µl and residual primers digested with Exonuclease I (Thermo Fisher) for 20 min at 37 °C.

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    Thermo Fisher exonuclease i
    The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.
    Exonuclease I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease i/product/Thermo Fisher
    Average 99 stars, based on 261 article reviews
    Price from $9.99 to $1999.99
    exonuclease i - by Bioz Stars, 2019-12
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    83
    Thermo Fisher trex1
    Expression and detection of <t>TREX1</t> proteins. A. The rabbit anti-TREX1 Ab detects the recombinant TREX1 (rV235fs) as well as E. coli heat shock protein 70 (Hsp70) in the immunogen on Western blot (WB). B. On strips from the same WB, the anti-TREX1 Ab detects a band at ∼75 kDa in 293T cells (left panel) that is eliminated after preincubation with E. coli Hsp70. The bands at 37 and 32 kDa are unchanged. C. A schematic of TREX1 transcript variants with solid red bar for coding sequence, open red bar for untranslated regions, and red line for intron. Transcript variant 1 retains the intron and encodes a longer N-terminus. D. 293T cells transiently transfected with fluorescent protein (FP)-tagged wild-type TREX1 (green) for isoforms-A (left panel) and B (right panel). Nuclei are counterstained with TO-PRO-3 (blue). Scale bar represents 28 μm. E. Representative WB of TREX1 from 293T cells transfected with wild-type TREX1 isoforms-B (lane 1) and A (lane 2) for positive controls and HeLa cells transfected with TREX1 siRNA 1 (lanes 3 and 7) or 2 (lanes 4 and 8), negative control (NC1, lanes 5 and 9) as well as an untreated control (lane 6). Cell lysates were evaluated at 48 (lanes 3–5) and 72 (lanes 7–9) h after transfection with siRNA. Membrane was stripped and reprobed with anti-β-actin Ab as a loading control.
    Trex1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    trex1 - by Bioz Stars, 2019-12
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    Image Search Results


    The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.

    Journal: Nucleic Acids Research

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination

    doi: 10.1093/nar/gkv172

    Figure Lengend Snippet: The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.

    Article Snippet: In a total of 10 μl reaction volume, 60-mer ssDNA (24 μM) was mixed with Dmc1 (12 μM) in a buffer B (50 mM Tris-HCl pH 7.5, 70 mM NaCl, 2 mM MgCl2 , 2 mM ATP) and incubated for 10 min at 37 o C. The Hop2–Mnd1 complex (2.5 μM) was added and incubated for 10 min followed by the addition of 10 units of exonuclease I (Thermo Scientific, 20 units/μl).

    Techniques: Binding Assay, Incubation, Electrophoresis

    Mutations in  STN1  result in abnormal telomere phenotypes.  (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P

    Journal: The Journal of Experimental Medicine

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects

    doi: 10.1084/jem.20151618

    Figure Lengend Snippet: Mutations in STN1 result in abnormal telomere phenotypes. (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P

    Article Snippet: Exonuclease I treatment was performed overnight at 37°C with 200 U exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation.

    Techniques: Hybridization, Fluorescence In Situ Hybridization

    Expression and detection of TREX1 proteins. A. The rabbit anti-TREX1 Ab detects the recombinant TREX1 (rV235fs) as well as E. coli heat shock protein 70 (Hsp70) in the immunogen on Western blot (WB). B. On strips from the same WB, the anti-TREX1 Ab detects a band at ∼75 kDa in 293T cells (left panel) that is eliminated after preincubation with E. coli Hsp70. The bands at 37 and 32 kDa are unchanged. C. A schematic of TREX1 transcript variants with solid red bar for coding sequence, open red bar for untranslated regions, and red line for intron. Transcript variant 1 retains the intron and encodes a longer N-terminus. D. 293T cells transiently transfected with fluorescent protein (FP)-tagged wild-type TREX1 (green) for isoforms-A (left panel) and B (right panel). Nuclei are counterstained with TO-PRO-3 (blue). Scale bar represents 28 μm. E. Representative WB of TREX1 from 293T cells transfected with wild-type TREX1 isoforms-B (lane 1) and A (lane 2) for positive controls and HeLa cells transfected with TREX1 siRNA 1 (lanes 3 and 7) or 2 (lanes 4 and 8), negative control (NC1, lanes 5 and 9) as well as an untreated control (lane 6). Cell lysates were evaluated at 48 (lanes 3–5) and 72 (lanes 7–9) h after transfection with siRNA. Membrane was stripped and reprobed with anti-β-actin Ab as a loading control.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: Expression and detection of TREX1 proteins. A. The rabbit anti-TREX1 Ab detects the recombinant TREX1 (rV235fs) as well as E. coli heat shock protein 70 (Hsp70) in the immunogen on Western blot (WB). B. On strips from the same WB, the anti-TREX1 Ab detects a band at ∼75 kDa in 293T cells (left panel) that is eliminated after preincubation with E. coli Hsp70. The bands at 37 and 32 kDa are unchanged. C. A schematic of TREX1 transcript variants with solid red bar for coding sequence, open red bar for untranslated regions, and red line for intron. Transcript variant 1 retains the intron and encodes a longer N-terminus. D. 293T cells transiently transfected with fluorescent protein (FP)-tagged wild-type TREX1 (green) for isoforms-A (left panel) and B (right panel). Nuclei are counterstained with TO-PRO-3 (blue). Scale bar represents 28 μm. E. Representative WB of TREX1 from 293T cells transfected with wild-type TREX1 isoforms-B (lane 1) and A (lane 2) for positive controls and HeLa cells transfected with TREX1 siRNA 1 (lanes 3 and 7) or 2 (lanes 4 and 8), negative control (NC1, lanes 5 and 9) as well as an untreated control (lane 6). Cell lysates were evaluated at 48 (lanes 3–5) and 72 (lanes 7–9) h after transfection with siRNA. Membrane was stripped and reprobed with anti-β-actin Ab as a loading control.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Expressing, Recombinant, Western Blot, Sequencing, Variant Assay, Transfection, Negative Control

    TREX1 positive cells are microglia and/or macrophages. A. Dual staining of frozen tissue from a case of RVCL with anti-MHC II (top panel, green) and anti-CD68 (bottom panel, green), microglial markers, and anti-TREX1 (red). Nuclei are counterstained with TO-PRO-3 (blue). Scale bar represents 28 μm. B. Two examples (upper and lower panels) of dual staining of formalin-fixed, paraffin-embedded human brain tissue from a normal control with RCA-1 (left panel, green), a microglial/macrophage and endothelial cell marker, and anti-TREX1 (red). Nuclei are counterstained with TO-PRO-3 (blue). Some cells stained with RCA-1 but not TREX1 are endothelial cells based on their morphology. Scale bars represent 28 μm on top panel and 14 μm on bottom panel.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: TREX1 positive cells are microglia and/or macrophages. A. Dual staining of frozen tissue from a case of RVCL with anti-MHC II (top panel, green) and anti-CD68 (bottom panel, green), microglial markers, and anti-TREX1 (red). Nuclei are counterstained with TO-PRO-3 (blue). Scale bar represents 28 μm. B. Two examples (upper and lower panels) of dual staining of formalin-fixed, paraffin-embedded human brain tissue from a normal control with RCA-1 (left panel, green), a microglial/macrophage and endothelial cell marker, and anti-TREX1 (red). Nuclei are counterstained with TO-PRO-3 (blue). Some cells stained with RCA-1 but not TREX1 are endothelial cells based on their morphology. Scale bars represent 28 μm on top panel and 14 μm on bottom panel.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Staining, Formalin-fixed Paraffin-Embedded, Marker

    Distribution of TREX1 expressing cells. A. Distribution of TREX1 + microglia by brain regions in normal controls and cases of RVCL and ischemic stroke. The proportion of TREX1 cells is shown relative to the mean number of microglia (Iba1 stained cells) for specific areas of the brain in normal controls (blue bars) and cases with RVCL (red bars) or ischemic stroke (green bars). Sections quantified in RVCL and ischemic stroke were taken from undamaged tissue. The number of tissue sections included for each observation is noted above the appropriate column. White matter (WM); Gray matter (GM). Data are presented as mean ± SEM. B-C. Distribution of TREX1 and Iba1 staining in the cerebral cortex of normal controls and cases of RVCL and ischemic stroke. The proportion of Iba1 (E) or TREX1 (F) cells located in the white matter for the frontal lobe, occipital lobe and the cerebral cortex in normal controls (blue bars) and cases with RVCL (red bars) or ischemic stroke (green bars). The values represent the mean among each group for cell counts in the white matter divided by the total cell counts in the white and gray matter for the specified region. Sections quantified in RVCL and ischemic stroke were taken from undamaged tissue. The cerebral cortex includes sections from the frontal, parietal, temporal and occipital lobes. The number of tissue sections included for each observation is noted above each column. Data are presented as mean ± SEM. D-E. Immunohistochemical staining of a normal control and subject with RVCL with Iba1 (left panels) and TREX1 (right panels) in the gray (top panels) and white matter (bottom panels). For RVCL, undamaged brain tissue is shown. Staining for Iba1 and TREX1 is developed with DAB (brown). Nuclei are counterstained with hematoxylin (blue). Scale bars represent 100 μm. F. Representative immunohistochemical staining of normal control gray matter with TREX1 microglia (DAB, brown) in association with microvasculature (arrows). Nuclei are counterstained with hematoxylin (blue). Scale bar represents 40 μm.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: Distribution of TREX1 expressing cells. A. Distribution of TREX1 + microglia by brain regions in normal controls and cases of RVCL and ischemic stroke. The proportion of TREX1 cells is shown relative to the mean number of microglia (Iba1 stained cells) for specific areas of the brain in normal controls (blue bars) and cases with RVCL (red bars) or ischemic stroke (green bars). Sections quantified in RVCL and ischemic stroke were taken from undamaged tissue. The number of tissue sections included for each observation is noted above the appropriate column. White matter (WM); Gray matter (GM). Data are presented as mean ± SEM. B-C. Distribution of TREX1 and Iba1 staining in the cerebral cortex of normal controls and cases of RVCL and ischemic stroke. The proportion of Iba1 (E) or TREX1 (F) cells located in the white matter for the frontal lobe, occipital lobe and the cerebral cortex in normal controls (blue bars) and cases with RVCL (red bars) or ischemic stroke (green bars). The values represent the mean among each group for cell counts in the white matter divided by the total cell counts in the white and gray matter for the specified region. Sections quantified in RVCL and ischemic stroke were taken from undamaged tissue. The cerebral cortex includes sections from the frontal, parietal, temporal and occipital lobes. The number of tissue sections included for each observation is noted above each column. Data are presented as mean ± SEM. D-E. Immunohistochemical staining of a normal control and subject with RVCL with Iba1 (left panels) and TREX1 (right panels) in the gray (top panels) and white matter (bottom panels). For RVCL, undamaged brain tissue is shown. Staining for Iba1 and TREX1 is developed with DAB (brown). Nuclei are counterstained with hematoxylin (blue). Scale bars represent 100 μm. F. Representative immunohistochemical staining of normal control gray matter with TREX1 microglia (DAB, brown) in association with microvasculature (arrows). Nuclei are counterstained with hematoxylin (blue). Scale bar represents 40 μm.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Expressing, Staining, Immunohistochemistry

    Specificity of rabbit pAb to TREX1. A. Immunofluorescent staining of 293T cells transfected with fluorescent protein-tagged wild-type TREX1 (FP-TREX, green) using the rabbit pAb to TREX1 (red) shows that the signals overlap (yellow). Scale bar represents 28 μm. B. Immunohistochemical staining of human brain tissue with rabbit pAb to TREX1 alone (left panel) and after pre-incubation of the Ab with E. coli heat shock protein 70 (Hsp70, right panel). Scale bar represents 100 μm. C. Immunohistochemical staining of human brain tissue with secondary Ab alone (left panel) or rabbit pAb to TREX1 (right panel). Scale bar represents 40 μm.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: Specificity of rabbit pAb to TREX1. A. Immunofluorescent staining of 293T cells transfected with fluorescent protein-tagged wild-type TREX1 (FP-TREX, green) using the rabbit pAb to TREX1 (red) shows that the signals overlap (yellow). Scale bar represents 28 μm. B. Immunohistochemical staining of human brain tissue with rabbit pAb to TREX1 alone (left panel) and after pre-incubation of the Ab with E. coli heat shock protein 70 (Hsp70, right panel). Scale bar represents 100 μm. C. Immunohistochemical staining of human brain tissue with secondary Ab alone (left panel) or rabbit pAb to TREX1 (right panel). Scale bar represents 40 μm.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Staining, Transfection, Immunohistochemistry, Incubation

    Endogenous expression of TREX1 in normal controls and heterozygous carriers of the V235fs mutant. A. Representative Western blot of transfected TREX1 in 293T cells (lanes 1–2, 9–10) and untransfected EBV transformed lymphocytes from normal controls and RVCL mutation carriers. The transfected protein is detected for both isoforms of wild-type (WT, lanes 1 and 9) and the frame-shift mutant of isoform-B (V235fs, lane 2). The frame-shift mutant of isoform-A (V290fs, lane 10) is not well seen. Both the wild-type and frame-shift mutant of isoform-A also produce the smaller isoform-B (*). Endogenous WT TREX1 is detected in all EBV transformed lymphocytes. The 37 kDa band represents TREX1 isoform-A (lanes 3–8). The V235fs mutant is detected in cell lines derived from heterozygous carriers of the mutation (lanes 4, 6, and 8) but not in normal controls (lanes 3, 5, and 7). Endogenous expression of the V290fs mutant is not detected. Membrane was stripped and reprobed with anti-β-actin as a loading control. B. The expression of TREX1 protein was normalized to a β-actin loading control. For each group, Control WT, Carrier WT and Carrier V235fs, data are shown for the 3 individuals analyzed followed by the average for that group. TREX1 levels are given relative to the average of WT in the 3 normal controls (Average Control), which was arbitrarily set to 1.0. Data are presented as mean ± SD and represent 8 independent samples.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: Endogenous expression of TREX1 in normal controls and heterozygous carriers of the V235fs mutant. A. Representative Western blot of transfected TREX1 in 293T cells (lanes 1–2, 9–10) and untransfected EBV transformed lymphocytes from normal controls and RVCL mutation carriers. The transfected protein is detected for both isoforms of wild-type (WT, lanes 1 and 9) and the frame-shift mutant of isoform-B (V235fs, lane 2). The frame-shift mutant of isoform-A (V290fs, lane 10) is not well seen. Both the wild-type and frame-shift mutant of isoform-A also produce the smaller isoform-B (*). Endogenous WT TREX1 is detected in all EBV transformed lymphocytes. The 37 kDa band represents TREX1 isoform-A (lanes 3–8). The V235fs mutant is detected in cell lines derived from heterozygous carriers of the mutation (lanes 4, 6, and 8) but not in normal controls (lanes 3, 5, and 7). Endogenous expression of the V290fs mutant is not detected. Membrane was stripped and reprobed with anti-β-actin as a loading control. B. The expression of TREX1 protein was normalized to a β-actin loading control. For each group, Control WT, Carrier WT and Carrier V235fs, data are shown for the 3 individuals analyzed followed by the average for that group. TREX1 levels are given relative to the average of WT in the 3 normal controls (Average Control), which was arbitrarily set to 1.0. Data are presented as mean ± SD and represent 8 independent samples.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Transformation Assay, Derivative Assay

    Immunohistochemical staining in white matter lesions with anti-TREX1. A. Staining for Iba1 and TREX1 in white matter lesions from cases with RVCL (left) or ischemic stroke (right). Representative images from chronic lesions are shown (400x magnification) with each successive image taken from two high-powered fields away. Staining for Iba1 and TREX1 is developed with DAB (brown). Nuclei are counterstained with hematoxylin (blue). Scale bar represents 100 μm. B. Panoramic view of the morphological changes in TREX1 + cells around a chronic lesion in ischemic stroke (right side) and their tracking in the adjacent white matter (WM) with relative absence of TREX1 + cells in the gray matter (GM). Similar findings are noted in RVCL lesions. Scale bar represents 1 mm.

    Journal: Brain pathology (Zurich, Switzerland)

    Article Title: TREX1 is expressed by microglia in normal human brain and increases in regions affected by ischemia

    doi: 10.1111/bpa.12626

    Figure Lengend Snippet: Immunohistochemical staining in white matter lesions with anti-TREX1. A. Staining for Iba1 and TREX1 in white matter lesions from cases with RVCL (left) or ischemic stroke (right). Representative images from chronic lesions are shown (400x magnification) with each successive image taken from two high-powered fields away. Staining for Iba1 and TREX1 is developed with DAB (brown). Nuclei are counterstained with hematoxylin (blue). Scale bar represents 100 μm. B. Panoramic view of the morphological changes in TREX1 + cells around a chronic lesion in ischemic stroke (right side) and their tracking in the adjacent white matter (WM) with relative absence of TREX1 + cells in the gray matter (GM). Similar findings are noted in RVCL lesions. Scale bar represents 1 mm.

    Article Snippet: The band at 37 kDa likely represents TREX1 transcript variant 1 (NM_016381.5) encoding isoform-A (NP_057465.1, 369 amino acids), which is included in NCBI databases.

    Techniques: Immunohistochemistry, Staining