e coli rnase h  (New England Biolabs)


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    Structured Review

    New England Biolabs e coli rnase h
    E Coli Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer). .. Fluorescence polarization assay The following fluorescein-labeled oligonucleotides were used: MHO-1_FL ( 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’ ); MHO-7_FL ( 5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’ ); MHO-6_FL ( 5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’ ).

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The WCEs were collected by centrifugation at 3,274 rcf for 5 min at 4°C, transferred into fresh pre-cooled 1.5 ml Eppendorf tubes and cleared by two consecutive rounds of centrifugation at 16,100 rcf at 4°C for 2 and 10 min, respectively. .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Amplification:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated. .. Finally, the resulting cDNA was amplified by PCR using oligo(dT) and oligo 9 primers, and sequenced.

    Article Title: RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
    Article Snippet: After probe hybridization and washing with PBS, samples were treated in 50 μl final volume for either RNAseIII treatment: 1X RNAseIII buffer, 1.5 μl Shortcut RNaseIII (New England Biolabs, cat. M0245s), and 1X MnCL2 or RNAseH treatment: (1X RNAseH buffer, 1.5 μl RNAseH (New England Biolabs, cat. M0297S) at 37°C for 2 hr. .. Samples were then blocked with 2x PBT:WBR 1 hr then processed as per protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above).

    Construct:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: To prepare reactions for the RNase H digestion of the uORF constructs, 750 μg of total protein in 300 μl of BB was incubated with 15 μl of 100 μM sequence-specific custom made oligos (MP7 and MP79) at 37°C for 10 min. (before adding the oligos we always set aside 5 % (37.5 μg) of total protein as ‘Input’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency of eIF3 subunits). .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Real-time Polymerase Chain Reaction:

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair
    Article Snippet: DNA was recovered with Chelex‐100. qPCR analysis was performed with enrichment calculated by ratio of DRIP/input. .. For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    Incubation:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Mapping of 3′ oligo 4–18S rRNA pairing 5 pmol of oligo 4 or VIC–oligo 4 were incubated with 30 pmol of 40S subunits purified from RRL and incubated for 30 min at 30°C in polysome buffer. .. Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated.

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer). .. Fluorescence polarization assay The following fluorescein-labeled oligonucleotides were used: MHO-1_FL ( 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’ ); MHO-7_FL ( 5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’ ); MHO-6_FL ( 5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’ ).

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair
    Article Snippet: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA. .. Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: To prepare reactions for the RNase H digestion of the uORF constructs, 750 μg of total protein in 300 μl of BB was incubated with 15 μl of 100 μM sequence-specific custom made oligos (MP7 and MP79) at 37°C for 10 min. (before adding the oligos we always set aside 5 % (37.5 μg) of total protein as ‘Input’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency of eIF3 subunits). .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: .. WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage). .. Reactions were stopped by the addition of stop buffer (20 mM Tris pH 7.9, 0.2% SDS, 50 mM EDTA, proteinase K) followed by incubation at 37°C for 10 min. After phenol/chloroform extraction and ethanol precipitation, samples were resuspended in 95% formamide buffer containing 5 mM EDTA and 0.1% SDS and were resolved by 15% denaturing PAGE.

    Western Blot:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: To prepare reactions for the RNase H digestion of the uORF constructs, 750 μg of total protein in 300 μl of BB was incubated with 15 μl of 100 μM sequence-specific custom made oligos (MP7 and MP79) at 37°C for 10 min. (before adding the oligos we always set aside 5 % (37.5 μg) of total protein as ‘Input’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency of eIF3 subunits). .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Recombinase Polymerase Amplification:

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer). .. Fluorescence polarization assay The following fluorescein-labeled oligonucleotides were used: MHO-1_FL ( 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’ ); MHO-7_FL ( 5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’ ); MHO-6_FL ( 5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’ ).

    Hybridization:

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer). .. Fluorescence polarization assay The following fluorescein-labeled oligonucleotides were used: MHO-1_FL ( 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’ ); MHO-7_FL ( 5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’ ); MHO-6_FL ( 5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’ ).

    Article Title: RNA from a simple-tandem repeat is required for sperm maturation and male fertility in Drosophila melanogaster
    Article Snippet: .. After probe hybridization and washing with PBS, samples were treated in 50 μl final volume for either RNAseIII treatment: 1X RNAseIII buffer, 1.5 μl Shortcut RNaseIII (New England Biolabs, cat. M0245s), and 1X MnCL2 or RNAseH treatment: (1X RNAseH buffer, 1.5 μl RNAseH (New England Biolabs, cat. M0297S) at 37°C for 2 hr. .. Samples were then blocked with 2x PBT:WBR 1 hr then processed as per protocol ‘TSA amplification for RNA-FISH probe detection’ (protocol three above).

    Flow Cytometry:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C. .. The next day we first set aside 5% of the flow through (FT) for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, and then we washed the beads three times in 1 ml BB still supplemented with the same concentration of the RNAse inhibitor as above); washed beads were always collected by centrifugations at 500 rcf for 2 min at 4°C.

    Protease Inhibitor:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The cell pellet was weighed and resuspended in the 1:1 ratio (ml:g) in the Breaking Buffer (BB) composed of only nano-pure water supplemented with the protease inhibitor complete EDTA-free tablet (Roche), 1 mM PMSF, 30 mM Imidazole and 0.486 mM β-mercaptoethanol (interestingly, the best reproducibility of our RaP-NiP results was achieved when we used only nano-pure water lacking all classical salt constituents). .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    Generated:

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: RNase protection assay DNA-RNA complexes were first generated by adding 20,000 cpm of 32 P-labelled MITF 1550–1740 RNA probe to separate tubes containing 2 μg of DNA oligonucleotides (MHO-1 or MHO-7) or 2 μg yeast tRNA as control. .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer).

    other:

    Article Title: Isolation and genome-wide characterization of cellular DNA:RNA triplex structures
    Article Snippet: RNAs that are associated via DNA-RNA heteroduplexes (R-loops) were digested with RNase H ( ).

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter
    Article Snippet: As additional support for the R-loop-mediated transcription blockage model, the blockage is not pronounced in the presence of RNase H during transcription, which removes R-loops and thus ‘unblocks’ DNA templates for further rounds of transcription.

    Article Title: Argonaute-based programmable RNase as a tool for cleavage of highly-structured RNA
    Article Snippet: Relative to cleavage of the unstructured target, RNase H only weakly cleaved all four sites, while DISC demonstrated more robust cleavage of TR6, TR8 and TR11 (Figure and ).

    Sequencing:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: To prepare reactions for the RNase H digestion of the uORF constructs, 750 μg of total protein in 300 μl of BB was incubated with 15 μl of 100 μM sequence-specific custom made oligos (MP7 and MP79) at 37°C for 10 min. (before adding the oligos we always set aside 5 % (37.5 μg) of total protein as ‘Input’ for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency of eIF3 subunits). .. The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    IF-P:

    Article Title: Strong transcription blockage mediated by R-loop formation within a G-rich homopurine–homopyrimidine sequence localized in the vicinity of the promoter
    Article Snippet: .. For example, if p = 0.2, the yield of full-size run-off transcript for R-loop-prone substrate in the presence of RNase H could reach up to 80% of the yield for non-R-loop-prone substrate. .. Thus, if the probability of R-loop formation during one round of transcription is sufficiently small, the yields of transcripts for R-loop-prone and non-R-loop-prone substrates in the presence of sufficiently high concentration of RNase H would be similar, which is observed in Figure (though more precise experiments would be needed to reliably estimate probability p ).

    Recombinant:

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair
    Article Snippet: For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA. .. Beads were incubated for 2.0 h at 37°C in the absence or presence of 15 μl of recombinant Escherichia coli RNase HI (75 units, NEB, M0297L), with shaking at 1,000 rpm (Eppendorf Thermomixer).

    Nucleic Acid Electrophoresis:

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex
    Article Snippet: For protein analysis, samples were digested with RNAse A and T1 for 1 h at 37°C before sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. .. Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C.

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: .. Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer). .. Fluorescence polarization assay The following fluorescein-labeled oligonucleotides were used: MHO-1_FL ( 5’-TAA AAA TCT CAA ATC AAG TTT C/36-FAM/-3’ ); MHO-7_FL ( 5’-ATT CAG TTC ACA GAT ACT GCA C/36-FAM/-3’ ); MHO-6_FL ( 5’-ATA CTG CAC ACT TAT TTG TAC A/36-FAM/-3’ ).

    In Vivo:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: Paragraph title: Yeast in vivo RNA–protein Ni2+ -pull down (RaP-NiP) assay ... The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C.

    RNA Sequencing Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. Another possibility is that the cell may target mRNAs with retained first-introns that form R-loops to be degraded by RNase H. In this model, mRNAs with retained introns that are observable by RNA-seq must have mechanisms to prevent the RNA from forming a long R-loop that would be susceptible to RNase H degradation. ..

    Pull Down Assay:

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C. .. Careful optimization of the RNase H reaction conditions using the uORF1-only construct revealed that 15 μl of 100 μM oligos and 10 U of RNase H works the best in achieving the efficient digestion of the uORF1 specific transcript in WCEs (750 μg of total protein) when incubated at 37°C for 20 min. For the Ni2+ -pull down assay, we first took 15 μl of the 50% Ni-sepharose slurry (GE Health Care) in a fresh Eppendorf tube and washed the beads in 1 ml of BB three times (washes were separated by centrifugations at 500 rcf for 2 min at 4°C).

    Magnetic Beads:

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair
    Article Snippet: The S9.6 antibody (2 μg) was coupled with 100 μl of protein G magnetic beads (Invitrogen). .. For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    Isolation:

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex
    Article Snippet: For 48S isolation, lysates were centrifuged on a 10–35% sucrose gradient at 45 000 rpm for 3 h in an SW50.1 rotor. .. Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C.

    Labeling:

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: In Vitro R-Loop Formation Experiments For R-loop formation experiments, the RNA oligonucleotide was either P32 -5′ end labeled or 5′-FAM labeled (FAM labeled oligo purchased from IDT) and then annealed to the DNA template strand (DNA oligo 1). .. WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage).

    Purification:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Mapping of 3′ oligo 4–18S rRNA pairing 5 pmol of oligo 4 or VIC–oligo 4 were incubated with 30 pmol of 40S subunits purified from RRL and incubated for 30 min at 30°C in polysome buffer. .. Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated.

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: .. WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage). .. Reactions were stopped by the addition of stop buffer (20 mM Tris pH 7.9, 0.2% SDS, 50 mM EDTA, proteinase K) followed by incubation at 37°C for 10 min. After phenol/chloroform extraction and ethanol precipitation, samples were resuspended in 95% formamide buffer containing 5 mM EDTA and 0.1% SDS and were resolved by 15% denaturing PAGE.

    Article Title: Engineering circular RNA for potent and stable translation in eukaryotic cells
    Article Snippet: .. Reactions were treated with RNase H (New England Biolabs) in the provided reaction buffer for 15 min at 37 C. RNA was column purified after digestion. .. Reverse transcription and cDNA synthesis For splice junction sequencing, splicing reactions enriched for circRNA with RNase R and then column purified were heated at 65 °C for 5 min and cooled on ice for 3 min to standardize secondary structure.

    Polymerase Chain Reaction:

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: Then, DNA–RNA hybrids were digested with 5 U of RNAse H (NEB) for 15 min at 37°C, extracted with phenol and ethanol precipitated. .. Finally, the resulting cDNA was amplified by PCR using oligo(dT) and oligo 9 primers, and sequenced.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage). .. Reactions were stopped by the addition of stop buffer (20 mM Tris pH 7.9, 0.2% SDS, 50 mM EDTA, proteinase K) followed by incubation at 37°C for 10 min. After phenol/chloroform extraction and ethanol precipitation, samples were resuspended in 95% formamide buffer containing 5 mM EDTA and 0.1% SDS and were resolved by 15% denaturing PAGE.

    Chromatin Immunoprecipitation:

    Article Title: RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair
    Article Snippet: Paragraph title: ChIP and DRIP ... For experiments with the RNase H treatment control, washed beads were re‐suspended in 300 μl of RNase H reaction buffer containing (NEB, M0297L) containing 4% glycerol and 20 mg/ml BSA.

    SDS Page:

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex
    Article Snippet: For protein analysis, samples were digested with RNAse A and T1 for 1 h at 37°C before sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. .. Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C.

    Rnase Protection Assay:

    Article Title: Characterizing the Coding Region Determinant-Binding Protein (CRD-BP)-Microphthalmia-associated Transcription Factor (MITF) mRNA interaction
    Article Snippet: Paragraph title: RNase protection assay ... Three 150 μl RNase H solutions containing 7.5 U of RNase H (New England Biolabs) in 1 X RNase H buffer were prepared and mixed with each hybridization reaction and incubated at 37°C for 1 hour, followed by heat inactivation at 65°C for 20 min. Digested RNA was then precipitated at -20°C for 1 hour after adding 225 μl of RNase Inactivation/precipitation III solution (Ambion RPA III kit), followed by 30 min centrifugation at 10,000 x g. RNA pellets were dried and suspended in 10 μl of Gel Loading Buffer II (RPA III kit) and fully denatured by heating at 95°C for 3 min. RNA samples were then resolved by gel electrophoresis on an 8% denaturing (7 M urea) polyacrylamide gel and exposed overnight to phosphor screen before visualization using the Cyclone Plus phosphorimager system (PerkinElmer).

    Agarose Gel Electrophoresis:

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex
    Article Snippet: Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C. .. Finally, the samples were analyzed by denaturing agarose gel electrophoresis, transferred to nitrocellulose membrane and exposed to X-ray films.

    In Vitro:

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: Paragraph title: In Vitro R-Loop Formation Experiments ... WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage).

    Ethanol Precipitation:

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability
    Article Snippet: WT-TFIIS and TFIISmut were added for 3 min, followed by addition of 5U of RNase H (NEB, M0297S) and incubated at RT for 30 min. For the experiment shown in E, the TEC/DNA scaffolds were purified via the biotin-tag on the non-transcribed strand after RNase H treatment to ensure that only anterior R-loops were detected (the label on posterior R-loops is released into the supernatant fraction due to RNase H cleavage). .. Reactions were stopped by the addition of stop buffer (20 mM Tris pH 7.9, 0.2% SDS, 50 mM EDTA, proteinase K) followed by incubation at 37°C for 10 min. After phenol/chloroform extraction and ethanol precipitation, samples were resuspended in 95% formamide buffer containing 5 mM EDTA and 0.1% SDS and were resolved by 15% denaturing PAGE.

    Concentration Assay:

    Article Title: Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq
    Article Snippet: .. In each of these final two wash steps, RNase H was added to a final concentration of 0.5 U/µl to the ‘RNase H’ treated sample, while dithiothreitol was added to a final concentration of 50 µM to the matched control sample. ..

    Article Title: In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation
    Article Snippet: The RNase H pre-reactions were then left at the room temperature for 10 min, after which 10 U of RNase H (NE Biolabs) was added for 20 min at 37°C. .. The next day we first set aside 5% of the flow through (FT) for western blot analysis of our routine check-ups of the Ni2+ -pull down efficiency, and then we washed the beads three times in 1 ml BB still supplemented with the same concentration of the RNAse inhibitor as above); washed beads were always collected by centrifugations at 500 rcf for 2 min at 4°C.

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    New England Biolabs e coli rnase h1
    <t>RNase</t> H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.
    E Coli Rnase H1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli rnase h1/product/New England Biolabs
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h1 - by Bioz Stars, 2020-02
    95/100 stars
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    95
    New England Biolabs e coli exoi
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    E Coli Exoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli exoi/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    e coli exoi - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    Image Search Results


    RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 deletion mutant is expressed in hepatocytes from knockout mice and is catalytically inactive. ( A ) Schematic of RNase H1 mRNA with LoxP sites indicated by asterisks, and schematic of deletion mutant mRNA missing exons 4 and 5. Arrows indicate the binding sites of PCR primers. The relative positions of the three catalytic amino acids D145, E186 and D210 are indicated by a, b and c, respectively. ( B ) PCR products and ( C ) RNase H1 activity in whole liver (WL), hepatocytes (Hep) and non-parenchymal cells (NP) from RNase H1 floxed mice (Flox), constitutive knockout (cKO) mice and inducible knockout (iKO) mice. Mice were sacrificed at 6 weeks of age. iKO mice were treated with tamoxifen for 1 week prior to sacrifice. WL, Hep and NP fractions were separated from one mouse liver. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Mutagenesis, Knock-Out, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Activity Assay

    Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Morphology and biochemical characteristics of hepatocyte mitochondria from RNase H1 knockout mice. Liver tissue was harvested from control RNase H1 floxed mice (flox + tam) and inducible RNase H1 knockout mice (iKO + tam) 1 week post tamoxifen treatment and control RNase H1 floxed mice (flox) and constitutive RNase H1 knockout mice (cKO) at weeks 6, 14 and 26. ( A ) Transmission electron micrograph. The white arrow indicates mitochondrial fusion and the black arrow indicates mitochondrial branching. * indicates glycogen appearance. ( B ) Western blots of proteins involved in mitochondrial fusion (OPA1) and fission (DRP1). The house-keeping gene GAPDH was used as a loading control for the western analysis. Each lane shows the expression levels of these proteins in individual mice ( N = 4). Also see Supplementary Figure S1D.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Mouse Assay, Transmission Assay, Western Blot, Expressing

    RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 expression and activity in constitutive RNase H1 knockout mice as a function of age. ( A ) RNase H1 mRNA expression levels in hepatocytes from RNase H1 floxed (open circles) and cKO mice (solid circles) were determined by qRT-PCR. ( B ) Cre mRNA expression levels in hepatocytes from cKO mice (solid circles) were determined by qRT-PCR. Data are the means of four animals per group, with the exception of week 8 ( n = 2) and week 26 ( n = 3). The error bars represent ± SEM. ( C ) Gel renaturation assay showing RNase H1 activity in hepatocytes from 6, 14 and 26 week old RNase H1 floxed (Flox) and cKO mice. Each lane is representative of hepatocytes RNase H activity from one mouse. The experiments were performed three times and representative results are shown.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Expressing, Activity Assay, Knock-Out, Mouse Assay, Quantitative RT-PCR

    RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: RNase H1 knockout impairs liver function. Plasma levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), total bilirubin (T. Bili), triglycerides and glucose were determined for cKO mice (solid circles, solid lines, N = 4), iKO mice treated with tamoxifen beginning at week 6 (solid boxes, dashed lines, N = 4), untreated floxed mice (open circles, solid lines, N = 4) and tamoxifen-treated floxed mice (open boxes, dashed lines, N = 4). The cKO mice were monitored from ages 6 to 26 weeks of age, and the iKO mice were continuously monitored 1 week prior to tamoxifen treatment, beginning at 6 weeks of age, until death or extreme illness, which occurred at 14 weeks of age. Data are the means of four animals and ± SEM. Also see Supplementary Figure S1. GraphPad two-way ANOVA calculations were used to determine significance. * (cKO) or # (iKO) P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, AST Assay, Mouse Assay

    ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: ASO activity impaired in RNase H1 knockout mice. ( A ) ASO activities in hepatocytes from RNase H1 floxed or knockout mice transfected with ASO at doses ranging from 40 pM to 250 nM. Hepatocytes transfected with ASO, and mRNA levels were evaluated 48-h post transfection. IC 50 correspond to the ASO concentrations resulting in 50% reduction of the target mRNA levels compared to the lipid-only treated cells. Data are the mean of triplicate determination and ± SEM. All changes between flox and knockout animals were significant, P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Allele-specific Oligonucleotide, Activity Assay, Knock-Out, Mouse Assay, Transfection

    Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Progression of mitochondrial dysfunction and liver disease absent functional RNase H1. Summary of the observations of RNase H1 iKO and cKO mice.

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Functional Assay, Mouse Assay

    Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Journal: Nucleic Acids Research

    Article Title: Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function

    doi: 10.1093/nar/gkw350

    Figure Lengend Snippet: Levels of mitochondrial DNA reduced and R-loop processing impaired in knockout livers. ( A ) Levels of mitochondrial DNA were determined by PCR. The liver tissue from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. The liver tissue from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( B ) S7 R-Loop levels determined in hepatocytes from the tamoxifen-treated RNase H1 floxed (hashed open bars) and iKO (hashed solid bars) mice were harvested 1 week after initiation of tamoxifen treatment. Hepatocytes from RNase H1 floxed (open bars) and cKO (solid bars) mice were harvested at 6, 14 and 26 weeks of age. Data are the means of four animals per group and error bars represent ±SEM. ( C ) S7 R-Loop levels determined after treatment of the hepatocyte samples from 6-week-old RNase H1 floxed (open bars) and cKO (solid bars) mice with 90 mg (high) or 18 mg (low) of E. coli RNase H1 prior to immunoprecipitation with the heteroduplex antibody. The figure shows results from a single experiment. GraphPad two-way ANOVA calculations were used to determine significance.* P

    Article Snippet: For samples spiked with RNase H1, 50 μg of total protein from sonicated lysate was incubated with 18 or 90 mg of E. coli RNase H1 (New England BioLabs), 3 mM MgCl2 and 1 × RNase H1 buffer (New England Biolabs) prior to immunoprecipitation.

    Techniques: Knock-Out, Polymerase Chain Reaction, Mouse Assay, Immunoprecipitation

    ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Journal: PLoS ONE

    Article Title: Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0101752

    Figure Lengend Snippet: ASO binding to naked 32 P-labeled SOD-1 minigene mRNA. Denaturing PAGE analysis of digestion reactions with ASOs and without ASO (labeled UTC). The bands corresponding to the RNase H1 cleavage products from on-target binding are labeled with ASO number in blue and off-target ASO cleavage products are labeled with red ASO numbers. The position of the off-target ASO hybridization in the SOD-1 minigene mRNA was determined by comparing the size of the off-target cleavage bands with the size of the on-target cleavage bands (joined by red lines).

    Article Snippet: To each reaction, 40 U of RNaseOUT (Life Technologies, P/N#100000840) and tris(2-carboxyethyl)phosphine) (TCEP; 0.2 mM final concentration) were added, and samples were incubated for 1 h. E. coli RNase H1 cleavage reactions were initiated by adding 25 U of enzyme (New England Biolabs) to a 100 µL reaction; samples were incubated at 37°C for 60 min.

    Techniques: Allele-specific Oligonucleotide, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Hybridization

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation