rnase v1 endonuclease protocol  (Thermo Fisher)


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    Name:
    Endonuclease V
    Description:
    Thermo Scientific Endonuclease V T maritima Endo V is a 3 endonuclease involved in DNA repair which initiates removal of deaminated bases from damaged DNA including uracil hypoxanthine and xanthine Endonuclease V is also active toward abasic sites and urea sites base pair mismatches flap and pseudo Y structures and small insertions deletions in DNA molecules The cleavage site generated by Endonuclease V is at the second phosphodiester bond 3 to a lesion Highlights• Optimal activity at temperatures of 65 to 70°CApplications• High throughput methods for mutation research• Studies in mutagenesis and DNA repair• Mismatch cleavage• GenotypingNoteUse of this enzyme in certain applications may be covered by patents and may require a license When the enzyme is in excess the primary nicked products experience a second nicking event on the complementary strand leading to a double stranded break At low concentrations however Endonuclease V first nicks a DNA strand at the lesions located closer to the 5 end of DNA molecule Single stranded DNA is cleaved with much lower efficiency Mg2 or Mn2 ions are required for enzyme activity
    Catalog Number:
    en0141
    Price:
    None
    Applications:
    Genotyping & Genomic Profiling|Rare & Low-Frequency Mutation Analysis|Gene Expression Analysis & Genotyping
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase v1 endonuclease protocol
    Thermo Scientific Endonuclease V T maritima Endo V is a 3 endonuclease involved in DNA repair which initiates removal of deaminated bases from damaged DNA including uracil hypoxanthine and xanthine Endonuclease V is also active toward abasic sites and urea sites base pair mismatches flap and pseudo Y structures and small insertions deletions in DNA molecules The cleavage site generated by Endonuclease V is at the second phosphodiester bond 3 to a lesion Highlights• Optimal activity at temperatures of 65 to 70°CApplications• High throughput methods for mutation research• Studies in mutagenesis and DNA repair• Mismatch cleavage• GenotypingNoteUse of this enzyme in certain applications may be covered by patents and may require a license When the enzyme is in excess the primary nicked products experience a second nicking event on the complementary strand leading to a double stranded break At low concentrations however Endonuclease V first nicks a DNA strand at the lesions located closer to the 5 end of DNA molecule Single stranded DNA is cleaved with much lower efficiency Mg2 or Mn2 ions are required for enzyme activity
    https://www.bioz.com/result/rnase v1 endonuclease protocol/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase v1 endonuclease protocol - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: TILLING to detect induced mutations in soybean
    Article Snippet: .. Genomic DNA restriction endonuclease digestion followed by high-throughput TILLING The gmrhg4 PCR product was amplified from the Forrest background and cloned using the pCR 4-TOPO TA kit (Invitrogen, Carlsbad, CA). .. Both strands of several clones were sequenced to generate consensus sequences for gmrhg4 and the homeolog/paralog.

    Transfection:

    Article Title: HMGB1 released by irradiated tumor cells promotes living tumor cell proliferation via paracrine effect
    Article Snippet: .. In detail, 293 T cells (1 × 106 cells per well; 6-well plate) were transfected with lentiviral construct expressing two gRNAs (gRNA1: 5′ GGAAGAGTCGACTCGCTT 3′, gRNA2: 5′ GTGATGTTGCGAAGAAAC 3′) and Cas9 endonuclease together with ecotropic packaging plasmids, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. .. Media containing viruses were collected 48 h after transfection and then utilized to infect HeLa and HT29 cells.

    Amplification:

    Article Title: TILLING to detect induced mutations in soybean
    Article Snippet: .. Genomic DNA restriction endonuclease digestion followed by high-throughput TILLING The gmrhg4 PCR product was amplified from the Forrest background and cloned using the pCR 4-TOPO TA kit (Invitrogen, Carlsbad, CA). .. Both strands of several clones were sequenced to generate consensus sequences for gmrhg4 and the homeolog/paralog.

    Mutagenesis:

    Article Title: Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes
    Article Snippet: .. In addition, cDNA generated from tmem88a mutants is not digested by ScaI endonuclease compared with wild type controls, providing further evidence that wild type transcripts are not present in tmem88a mutant embryos ( ). .. Reduced expression of some key cardiac and haematopoietic factors is observed in tmem88a mutant embryos Tmem88a knockdown has previously been shown to reduce expression of haematopoietic genes [ , ].

    Incubation:

    Article Title: Multiplex quantitative analysis of microRNA expression via exponential isothermal amplification and conformation-sensitive DNA separation
    Article Snippet: .. The cleavage and control reactions were incubated at 37 °C for 3 h. Bsm DNA polymerase, Buffer R, and Nb.Bpu10I nicking endonuclease were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). .. Exponential isothermal amplification Two reaction mixtures were prepared separately on ice.

    Article Title: Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity
    Article Snippet: .. The blots were incubated with a rabbit anti-endonuclease I antibody (diluted 1:1,000) overnight at 4°C and for 1 hr at room temperature with a goat anti-rabbit IgG, HRP-coupled secondary antibody (Invitrogen) (diluted 1:5,000). .. The westerns were developed with enhanced chemiluminescence (Bio-Rad).

    Article Title: A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity
    Article Snippet: .. Chemical cross-linking analysis The purified ARMAN-2 tRNA endonuclease (∼500 ng) was incubated in PBS buffer containing 50 mM NaCl with various concentrations (0–5 mM) of BS3 (Bis[sulfosuccinimidyl] suberate) (Thermo Scientific, Rockford, IL, USA) at room temperature for 30 min. .. Reactions were stopped by adding 1 M Tris–HCl (pH 8.0) and analyzed by 10–20% PAGE containing SDS.

    Construct:

    Article Title: HMGB1 released by irradiated tumor cells promotes living tumor cell proliferation via paracrine effect
    Article Snippet: .. In detail, 293 T cells (1 × 106 cells per well; 6-well plate) were transfected with lentiviral construct expressing two gRNAs (gRNA1: 5′ GGAAGAGTCGACTCGCTT 3′, gRNA2: 5′ GTGATGTTGCGAAGAAAC 3′) and Cas9 endonuclease together with ecotropic packaging plasmids, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. .. Media containing viruses were collected 48 h after transfection and then utilized to infect HeLa and HT29 cells.

    Purification:

    Article Title: A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity
    Article Snippet: .. Chemical cross-linking analysis The purified ARMAN-2 tRNA endonuclease (∼500 ng) was incubated in PBS buffer containing 50 mM NaCl with various concentrations (0–5 mM) of BS3 (Bis[sulfosuccinimidyl] suberate) (Thermo Scientific, Rockford, IL, USA) at room temperature for 30 min. .. Reactions were stopped by adding 1 M Tris–HCl (pH 8.0) and analyzed by 10–20% PAGE containing SDS.

    Polymerase Chain Reaction:

    Article Title: Diversity of Green-Like and Red-Like Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Large-Subunit Genes (cbbL) in Differently Managed Agricultural Soils
    Article Snippet: .. Ten microliters of each PCR product was hydrolyzed with 2 U of the restriction endonuclease BbvI (MBI Fermentas). .. Restriction fragments were analyzed in 3.5% (wt/vol) agarose gels (PeqLab) and visualized as described previously.

    Article Title: TILLING to detect induced mutations in soybean
    Article Snippet: .. Genomic DNA restriction endonuclease digestion followed by high-throughput TILLING The gmrhg4 PCR product was amplified from the Forrest background and cloned using the pCR 4-TOPO TA kit (Invitrogen, Carlsbad, CA). .. Both strands of several clones were sequenced to generate consensus sequences for gmrhg4 and the homeolog/paralog.

    Generated:

    Article Title: Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes
    Article Snippet: .. In addition, cDNA generated from tmem88a mutants is not digested by ScaI endonuclease compared with wild type controls, providing further evidence that wild type transcripts are not present in tmem88a mutant embryos ( ). .. Reduced expression of some key cardiac and haematopoietic factors is observed in tmem88a mutant embryos Tmem88a knockdown has previously been shown to reduce expression of haematopoietic genes [ , ].

    Activity Assay:

    Article Title: LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity
    Article Snippet: .. In all endonuclease activity assays commercial Eco RI restriction enzyme (Thermo Fisher Scientific) was used as control. .. Reactions were stopped by addition 1/10 volume of stop solution (50 mM EDTA pH 8, 50% glycerol, 0.02% orange G) and products were analyzed by electrophoresis in 1% agarose gels.

    Expressing:

    Article Title: HMGB1 released by irradiated tumor cells promotes living tumor cell proliferation via paracrine effect
    Article Snippet: .. In detail, 293 T cells (1 × 106 cells per well; 6-well plate) were transfected with lentiviral construct expressing two gRNAs (gRNA1: 5′ GGAAGAGTCGACTCGCTT 3′, gRNA2: 5′ GTGATGTTGCGAAGAAAC 3′) and Cas9 endonuclease together with ecotropic packaging plasmids, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. .. Media containing viruses were collected 48 h after transfection and then utilized to infect HeLa and HT29 cells.

    High Throughput Screening Assay:

    Article Title: TILLING to detect induced mutations in soybean
    Article Snippet: .. Genomic DNA restriction endonuclease digestion followed by high-throughput TILLING The gmrhg4 PCR product was amplified from the Forrest background and cloned using the pCR 4-TOPO TA kit (Invitrogen, Carlsbad, CA). .. Both strands of several clones were sequenced to generate consensus sequences for gmrhg4 and the homeolog/paralog.

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  • 92
    Thermo Fisher rnase v1
    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific <t>RNase</t> V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.
    Rnase V1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase v1/product/Thermo Fisher
    Average 92 stars, based on 123 article reviews
    Price from $9.99 to $1999.99
    rnase v1 - by Bioz Stars, 2020-07
    92/100 stars
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    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Journal: Nucleic Acids Research

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    doi: 10.1093/nar/gky073

    Figure Lengend Snippet: Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Article Snippet: RNase V1 probing used a final concentration of 0.01 U/μl (ThermoFisher Scientific, #AM2275) for 5 min at 37°C.

    Techniques: Sequencing

    Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Journal: Nucleic Acids Research

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    doi: 10.1093/nar/gky073

    Figure Lengend Snippet: Addition of CA-repeats does not change the secondary structure of the coat RBS stem–loop. ( A ) Enzymatic and chemical structure probing was conducted on control mRNA (00) or mRNAs with tails of 4, 6 or 8 (CA)-repeats, (see Materials and Methods), as indicated. The mRNAs were mock-treated (lanes ‘–’), partially digested with double-strand-specific RNase V1 (V1), or treated with lead(II) acetate (Pb 2+ ). UCGA: sequencing reactions on (CA)8 mRNA. The position of the SD and AUG start codon are indicated by red boxes. Regions of reactivity toward RNase V1 (red solid line) and lead(II) acetate (red dashed line) are indicated on the autoradiogram. ( B ) The localization of RNase V1 (filled triangles) and lead(II) acetate (black dots) cuts are shown on the secondary structure of the 5′-segment of (CA)8 mRNA. Black boxes: SD and AUG.

    Article Snippet: RNase V1 probing used a final concentration of 0.01 U/μl (ThermoFisher Scientific, #AM2275) for 5 min at 37°C.

    Techniques: Sequencing

    Enzymatic degradation of RNA causes protein aggregation. a Diagram showing the experimental design. b SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-). c Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg 2+ . Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1),

    Journal: bioRxiv

    Article Title: Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates

    doi: 10.1101/841577

    Figure Lengend Snippet: Enzymatic degradation of RNA causes protein aggregation. a Diagram showing the experimental design. b SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-). c Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg 2+ . Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1),

    Article Snippet: Enzymes and reagents RNase T1 (AM2280), RNase V1 (AM2275), RNase A/T1 cocktail (EN0551), DNase I (2222), and Yeast t-RNA (15401-011) were from Thermo Fisher Scientific.

    Techniques: SDS Page, Incubation

    Representative probing experiments using 5′-endlabeled aptamer A011 RNA and analyzed by ( A ) 20% or ( B ) 10% denaturing PAGE. Lanes 1 and 13, undigested RNA control (con.); lanes 2 and 14, limited alkaline hydrolysis; lanes 3, 12, 15 and 24, partial digestion with RNase T1 under denaturing (denat.) conditions; lanes 4, 5, 16 and 17, with two concentrations of RNase T1 under native conditions; lanes 6, 7, 18 and 19, with two concentrations of RNase V1; lanes 8, 9, 20 and 21, with two concentrations of nuclease P1; lane 10 and 22, with nuclease S1; lanes 11 and 23, Pb2+-induced hydrolysis. For experimental details, see Materials and Methods. Alkaline hydrolysis bands and the corresponding structure elements are indicated at the left and right margins, respectively, according to the numbering system presented in Figure 2 (A011, center). ( C , D ) Illustration of prominent cleavage sites in the context of the secondary structure of A011, derived from probing experiments such as those shown in panels A and B . Symbol sizes suggests the relative intensity of cleavage bands based on visual inspection.

    Journal: International Journal of Molecular Sciences

    Article Title: 2′-Fluoro-Pyrimidine-Modified RNA Aptamers Specific for Lipopolysaccharide Binding Protein (LBP)

    doi: 10.3390/ijms19123883

    Figure Lengend Snippet: Representative probing experiments using 5′-endlabeled aptamer A011 RNA and analyzed by ( A ) 20% or ( B ) 10% denaturing PAGE. Lanes 1 and 13, undigested RNA control (con.); lanes 2 and 14, limited alkaline hydrolysis; lanes 3, 12, 15 and 24, partial digestion with RNase T1 under denaturing (denat.) conditions; lanes 4, 5, 16 and 17, with two concentrations of RNase T1 under native conditions; lanes 6, 7, 18 and 19, with two concentrations of RNase V1; lanes 8, 9, 20 and 21, with two concentrations of nuclease P1; lane 10 and 22, with nuclease S1; lanes 11 and 23, Pb2+-induced hydrolysis. For experimental details, see Materials and Methods. Alkaline hydrolysis bands and the corresponding structure elements are indicated at the left and right margins, respectively, according to the numbering system presented in Figure 2 (A011, center). ( C , D ) Illustration of prominent cleavage sites in the context of the secondary structure of A011, derived from probing experiments such as those shown in panels A and B . Symbol sizes suggests the relative intensity of cleavage bands based on visual inspection.

    Article Snippet: Enzymatic digestions were performed as follows: RNase T1 (Thermo Fisher Scientific, Waltham, MA, USA) either in 20 mM sodium citrat (pH 5.0), 7 mM urea, 1 mM EDTA and 1 unit RNase T1 for 30 min at 55 °C (denaturing conditions) or in binding buffer (see above) with 0.5 or 0.05 units for 20 min at room temperature (RT); 0.01 or 0.05 units RNase V1 (Thermo Fisher Scientific Pierce) in binding buffer for 20 min at RT; 1 unit RNase S1 (Thermo Fisher Scientific, Waltham, MA, USA) in binding buffer containing 4.5 mM ZnSO4 for 20 min at RT; 0.01 or 0.05 units nuclease P1 (Sigma-Aldrich, St. Louis, MO, USA) in binding buffer containing 0.4 mM ZnSO4 for 20 min at RT.

    Techniques: Polyacrylamide Gel Electrophoresis, Derivative Assay