exonucleases iii  (Thermo Fisher)


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    Structured Review

    Thermo Fisher exonucleases iii
    Exonucleases Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonucleases iii/product/Thermo Fisher
    Average 88 stars, based on 1 article reviews
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    exonucleases iii - by Bioz Stars, 2020-09
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    Article Title: Characterization of MHC Class I alleles in sooty mangabeys as a tool for evaluating cellular immunity in natural hosts of SIV infection
    Article Snippet: .. Briefly, double-stranded cDNA (dsDNA) was digested with Gene II and Exonucleases III (Gene Trapper cDNA Positive Selection System, Invitrogen) to obtain single-strand cDNA (ssDNA). .. The MHC class I specific oligonucleotide probe was biotinylated and used for hybridization of ssDNA.

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    Thermo Fisher exonuclease iii
    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with <t>RNase</t> A, DNase I, Exonuclease I (Exo I), Exonuclease <t>III</t> (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp trex2 mm04210320 m1
    DNA-driven gene expression signature is not induced in the tongue of the <t>Trex2</t> −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P
    Gene Exp Trex2 Mm04210320 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Journal: Nucleic Acids Research

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

    doi: 10.1093/nar/gkaa160

    Figure Lengend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Article Snippet: Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: DNA extraction and characterization L- and S-EVs were treated with rDNase I (2 U/μl, DNA-Free Kit, Ambion) and Exonuclease III (200 U/μl, Thermofisher).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    Expression of DIRS-1 transcripts in different D. discoideum knock-out strains. A ) consisting of long inverted terminal repeats ( left ITR (330 bp) and right ITR (358 bp)) that flank three open reading frames. ORF1 encodes a putative Gag protein, ORF2 encodes a tyrosine recombinase ( YR ), and ORF3 encodes the pol gene, which comprises a RT, an RNase H ( RH ), and a methyltransferase ( MT ) domain. The internal complementary region ( ICR ) next to the right ITR exhibits sequences that are inverse complementary to the left and the right ITRs. A1 and A2 indicate amplicons used for qRT-PCR. P1 to P3 show the binding positions of the radioactively labeled oligonucleotides. Oligonucleotide probes ( P ) shown above the element are directed against sense transcripts. B , Northern blot analysis of DIRS-1 sense transcripts in deletion strains and the Ax2 wild type. Three 32 P-labeled oligonucleotides (P1–P3) were used to detect sense transcripts. As a loading control, the membrane was rehybridized with a 32 P-labeled oligonucleotide probe directed against the actin mRNA. C , relative expression of DIRS-1 transcripts (sense and antisense) in the indicated deletion strains and the Ax2 wild type measured by qRT-PCR using two different DIRS-1 amplicons ( A1 and A2 ) normalized to cinD mRNA. The data were plotted relative to DIRS-1 expression in Ax2 (transcript level is set to 100%). A1 , n = 8. Error bars : mean with S.D.; paired t test: p = 0.0001 (***) ( Ax2 wt : agnA−), p = 0.0037 (**) ( Ax2 : agnB−), p = 0.0010 (***) (Ax2: agnA−/agnB−). A2 , n = 4 (agnA−/agnB ko n = 4). Error bars : mean with S.D., paired t test: p =

    Journal: The Journal of Biological Chemistry

    Article Title: Argonaute Proteins Affect siRNA Levels and Accumulation of a Novel Extrachromosomal DNA from the Dictyostelium Retrotransposon DIRS-1 *

    doi: 10.1074/jbc.M114.612663

    Figure Lengend Snippet: Expression of DIRS-1 transcripts in different D. discoideum knock-out strains. A ) consisting of long inverted terminal repeats ( left ITR (330 bp) and right ITR (358 bp)) that flank three open reading frames. ORF1 encodes a putative Gag protein, ORF2 encodes a tyrosine recombinase ( YR ), and ORF3 encodes the pol gene, which comprises a RT, an RNase H ( RH ), and a methyltransferase ( MT ) domain. The internal complementary region ( ICR ) next to the right ITR exhibits sequences that are inverse complementary to the left and the right ITRs. A1 and A2 indicate amplicons used for qRT-PCR. P1 to P3 show the binding positions of the radioactively labeled oligonucleotides. Oligonucleotide probes ( P ) shown above the element are directed against sense transcripts. B , Northern blot analysis of DIRS-1 sense transcripts in deletion strains and the Ax2 wild type. Three 32 P-labeled oligonucleotides (P1–P3) were used to detect sense transcripts. As a loading control, the membrane was rehybridized with a 32 P-labeled oligonucleotide probe directed against the actin mRNA. C , relative expression of DIRS-1 transcripts (sense and antisense) in the indicated deletion strains and the Ax2 wild type measured by qRT-PCR using two different DIRS-1 amplicons ( A1 and A2 ) normalized to cinD mRNA. The data were plotted relative to DIRS-1 expression in Ax2 (transcript level is set to 100%). A1 , n = 8. Error bars : mean with S.D.; paired t test: p = 0.0001 (***) ( Ax2 wt : agnA−), p = 0.0037 (**) ( Ax2 : agnB−), p = 0.0010 (***) (Ax2: agnA−/agnB−). A2 , n = 4 (agnA−/agnB ko n = 4). Error bars : mean with S.D., paired t test: p =

    Article Snippet: 10 μl of cytoplasmic DNA from the agnA− strain were subjected to DNase I, exonuclease III, exonuclease I, S1 nuclease and micrococcal nuclease (Thermo Fisher) in a final volume of 20 μl according to the manufacturer's instructions.

    Techniques: Expressing, Knock-Out, Quantitative RT-PCR, Binding Assay, Labeling, Northern Blot

    DNA-driven gene expression signature is not induced in the tongue of the Trex2 −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: DNA-driven gene expression signature is not induced in the tongue of the Trex2 −/− Dnase1l2 −/− mice. Expression of the indicated genes in the tongue from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice, as determined by RT-qPCR. Each dot indicates a sample from an individual mouse, and horizontal lines represent the mean value. The results from one of two independent experiments with similar results are shown. Significant differences, by the Mann-Whitney test between genotypes: *P

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Expressing, Mouse Assay, Quantitative RT-PCR, MANN-WHITNEY

    Double Trex2 and Dnase1l2 deficiency does not trigger parakeratosis in the skin. H E (lanes 1 and 2) staining and Hoechst DNA labelling (lanes 3 and 4) in back skin and tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back skin samples were from 6 days-old mice which are consistently in the anagen phase of the hair cycle. Likewise, adult back skin was free from parakeratosis in all samples investigated. Tongue samples were from adult 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: Double Trex2 and Dnase1l2 deficiency does not trigger parakeratosis in the skin. H E (lanes 1 and 2) staining and Hoechst DNA labelling (lanes 3 and 4) in back skin and tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back skin samples were from 6 days-old mice which are consistently in the anagen phase of the hair cycle. Likewise, adult back skin was free from parakeratosis in all samples investigated. Tongue samples were from adult 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Staining, Mouse Assay

    Double Trex2 and Dnase1l2 deficiency leads to an increase of fragmented DNA in the upper keratinocyte layers of the tongue, but not of the epidermis. TUNEL labelling of free 3′-OH DNA ends (red) and nuclei (blue) counterstained with Hoechst of back and ear skin, and the filiform papillae and back side tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back and ear skin samples were from 6 days-old mice. Tongue samples were from 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: Double Trex2 and Dnase1l2 deficiency leads to an increase of fragmented DNA in the upper keratinocyte layers of the tongue, but not of the epidermis. TUNEL labelling of free 3′-OH DNA ends (red) and nuclei (blue) counterstained with Hoechst of back and ear skin, and the filiform papillae and back side tongue sections from wt, Trex2 −/− , Dnase1l2 −/− and Trex2 −/− Dnase1l2 −/− mice. Back and ear skin samples were from 6 days-old mice. Tongue samples were from 7–9 weeks-old mice. The images are representative for at least five mice from each genotype. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: TUNEL Assay, Mouse Assay

    The nucleases Trex2 and DNase1L2 are mostly expressed in the keratinocyte upper layers of the skin and tongue epithelia. ( a ) Immunofluorescence images showing DNase1L2 (red) and DNA (blue) staining in the back skin and tongue sections from wild-type (wt) and Dnase1l2 knockout ( Dnase1l2 −/− ) mice. ( b ) Immunofluorescence images illustrating Trex2 (red) and DNA (blue) staining in back skin and tongue sections from wt and Trex2 knockout ( Trex2 −/− ) mice. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Journal: Scientific Reports

    Article Title: Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses

    doi: 10.1038/s41598-017-12308-4

    Figure Lengend Snippet: The nucleases Trex2 and DNase1L2 are mostly expressed in the keratinocyte upper layers of the skin and tongue epithelia. ( a ) Immunofluorescence images showing DNase1L2 (red) and DNA (blue) staining in the back skin and tongue sections from wild-type (wt) and Dnase1l2 knockout ( Dnase1l2 −/− ) mice. ( b ) Immunofluorescence images illustrating Trex2 (red) and DNA (blue) staining in back skin and tongue sections from wt and Trex2 knockout ( Trex2 −/− ) mice. Scale bars = 25 μm. Dashed lines, epidermal-dermal border; continuous lines, bottom of the stratum corneum; dotted lines, upper border of the stratum corneum.

    Article Snippet: TaqMan Gene Expression Master Mix and predesigned probes (Applied Biosystems) were used to quantify mRNA expression of the following genes: Trex2 (Mm04210320_m1), Dnase1l2 (Mm00481868_g1), Ifnβ (Mm00439552_s1), Tnf α (Mm00443258_m1), Cxcl10 (Mm00445235_m1), Mx1 (Mm00487796_m1), Aim2 (Mm01295719_m1), IL6 (Mm00446190_m1), IL1β (Mm00434228_m1), and Sdha (Mm01352366_m1).

    Techniques: Immunofluorescence, Staining, Knock-Out, Mouse Assay