mnase reaction buffer  (Thermo Fisher)


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    Name:
    Reaction Buffer
    Description:
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    Catalog Number:
    b43
    Price:
    None
    Applications:
    In Vitro Transcription|One-Step qRT-PCR|PCR & Real-Time PCR|RT-PCR|Real Time PCR (qPCR)|Reverse Transcription|Two-Step RT-PCR|Gene Expression Analysis & Genotyping
    Category:
    Lab Reagents and Chemicals
    Buy from Supplier


    Structured Review

    Thermo Fisher mnase reaction buffer
    Thermo Scientific 10X Reaction Buffer with MgCl2 is used with RNase free DNase I an endonuclease that digests single and double stranded DNA
    https://www.bioz.com/result/mnase reaction buffer/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mnase reaction buffer - by Bioz Stars, 2020-05
    99/100 stars

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    Related Articles

    Amplification:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. One microliter of the RT mix (3× VILO Reaction Mix (Thermo Fisher) and 3× SuperScript Enzyme Mix (Thermo Fisher) in RNase-free water) was added to the sample plate and incubated at 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. For single-cell RT-qPCR in Supplementary Fig. , the RT product was diluted 1:5 in RNase-free water for qPCR analysis.

    Labeling:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Purification:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    SYBR Green Assay:

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Concentration Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Incubation:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

    Article Title: Calpain and PARP Activation during Photoreceptor Cell Death in P23H and S334ter Rhodopsin Mutant Rats
    Article Snippet: .. Briefly, unfixed cryosections were incubated for 15 min in calpain reaction buffer (CRB; 25 mM HEPES, 65 mM KCl, 2 mM MgCl2 , 1,5 mM CaCl2 , 2 mM DTT) and then sections were incubated at 35°C for 1 h in the dark in 2 µM fluorescent calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl- L-methionine amide (CMAC, t-BOC-Leu-Met; Molecular Probes, Inc. Eugene, USA). .. Fluorescence was generated by calpain-dependent cleavage of t-Boc-Leu-Met-CMAC.

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. One microliter of the RT mix (3× VILO Reaction Mix (Thermo Fisher) and 3× SuperScript Enzyme Mix (Thermo Fisher) in RNase-free water) was added to the sample plate and incubated at 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. For single-cell RT-qPCR in Supplementary Fig. , the RT product was diluted 1:5 in RNase-free water for qPCR analysis.

    Plasmid Preparation:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    Recombinant:

    Article Title: TDP1 promotes assembly of non-homologous end joining protein complexes on DNA
    Article Snippet: .. Purified, recombinant his-tagged TDP1 was combined with 25 fmol of 32 P labeled 3′ biotin-adduct substrate in 10 μl of TDP1 reaction buffer (50 mM Tris-Cl, pH 8.5, 100 mM NaCl, 25 μg/ml BSA, 0.5 mM EDTA) with 1% PVA, incubated at 37° C for 15 min and the reaction was stopped by addition of 10 μl of 2x TBE-Urea Sample Buffer (Invitrogen). ..

    Polymerase Chain Reaction:

    Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
    Article Snippet: .. For RT–PCR, ∼5 μg of total RNA were treated with 5 units of RNase-free DNase (Boehringer-Mannheim) in 1× PCR buffer (GIBCO-BRL) containing 2.5 m m MgCl2 at 37°C for 30 min. After heat inactivation at 80°C for 5 min, samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with ethanol. .. The RNA was reverse transcribed using 5 pmoles of random hexamers (Pharmacia Biotech) in a 12-μl reaction containing 1× PCR buffer (GIBCO-BRL), 2.1 m m MgCl2 , 0.5 m m of each deoxynucleotide triphosphate (dNTP), 10 m m dithiothreitol, and 120 units of Superscript II reverse transcriptase (GIBCO-BRL) by incubating at 25°C for 10 min followed by 42°C for 55 min and heat inactivation at 70°C for 20 min.

    Article Title: HoxC5 and miR-615-3p target newly evolved genomic regions to repress hTERT and inhibit tumorigenesis
    Article Snippet: .. The PCR reaction mixture contained 1X TRAP buffer, 4 ng/μl ACX primer 5′-GCGCGGCTTA CCCTTACCCTTACCCTAACC-3′, 15% glycerol, 1:2000 SYBR Green from Invitrogen, 0.08 U/μl Taq polymerase and was initiated at 94 °C for 2 min, followed by 45 cycles of 95 °C for 5 s, 50 °C for 6 s, and 72 °C for 10 s. The threshold cycle values (Ct) were determined from semi log amplification plots. .. Genomic DNA was extracted using Gentra Puregene Genomic DNA Purification Kit (Qiagen).

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs
    Article Snippet: .. Next, 0.5 μL of genomic DNA digestion mix (0.1 U of DNase I Amplification Grade (Thermo Fisher) and 2× DNase I Reaction Buffer (Thermo Fisher) in RNase-free water) was added to 1 μL of the single-cell lysate in a 96-well PCR plate and incubated at 25 °C for 5 min. After genomic DNA digestion, we added 0.5 µL of denaturing mix (8 mM EDTA and 0.02% NP40 in RNase-free water) to the digested sample, followed by incubation at 70 °C for 5 min to inactivate DNase I and desaturate the RNAs. .. One microliter of the RT mix (3× VILO Reaction Mix (Thermo Fisher) and 3× SuperScript Enzyme Mix (Thermo Fisher) in RNase-free water) was added to the sample plate and incubated at 25 °C for 10 min, 42 °C for 60 min, and 85 °C for 5 min. For single-cell RT-qPCR in Supplementary Fig. , the RT product was diluted 1:5 in RNase-free water for qPCR analysis.

    Staining:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Binding Assay:

    Article Title: Rolling circle replication requires single-stranded DNA binding protein to avoid termination and production of double-stranded DNA
    Article Snippet: .. RCA of DNA plasmids and digestion of RCA products We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). .. We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions.

    SDS Page:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Molecular Weight:

    Article Title: Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    Article Snippet: .. Purified sortase A pentamutant or heptamutant stock solution (see Support Protocol 1) 10x sortase reaction buffer: pentamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl, 100 mM CaCl2 ), heptamutant (500 mM Tris-HCl, pH 7.5, 1.5 M NaCl or 10x phosphate buffered saline (PBS)) 1.5 mL microcentrifuge tubes 4 °C or 25 °C incubator Zeba desalting column (ThermoFisher) Centrifugal concentrator with MWCO below the molecular weight of target protein (Millipore) Additional reagents and equipment for SDS-PAGE, immunoblot and Coomassie stain. .. Purified LPXTG-containing target protein (cannot be in phosphate containing buffer when using pentamutant variant of sortase) Oligoglycine peptide probe stock solution (5–10 mM in DMSO or H2 O) Ni-NTA column ( optional ) Imidazole ( optional )

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity
    Article Snippet: .. For RT–PCR, ∼5 μg of total RNA were treated with 5 units of RNase-free DNase (Boehringer-Mannheim) in 1× PCR buffer (GIBCO-BRL) containing 2.5 m m MgCl2 at 37°C for 30 min. After heat inactivation at 80°C for 5 min, samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and precipitated with ethanol. .. The RNA was reverse transcribed using 5 pmoles of random hexamers (Pharmacia Biotech) in a 12-μl reaction containing 1× PCR buffer (GIBCO-BRL), 2.1 m m MgCl2 , 0.5 m m of each deoxynucleotide triphosphate (dNTP), 10 m m dithiothreitol, and 120 units of Superscript II reverse transcriptase (GIBCO-BRL) by incubating at 25°C for 10 min followed by 42°C for 55 min and heat inactivation at 70°C for 20 min.

    Article Title: Collagen I Induces Discoidin Domain Receptor (DDR) 1 Expression through DDR2 and a JAK2-ERK1/2-mediated Mechanism in Primary Human Lung Fibroblasts *
    Article Snippet: .. For reverse transcription, 100 ng of total RNA was added to 50 μl of reaction buffer containing RT-PCR buffer, MgCl2 (5.5 m m ), desoxyribonucleoside triphosphate mixture (500 μ m ), random hexamers (2.5 μ m ), RNase inhibitors (0.4 unit/μl), and MultiScribe reverse transcriptase (1.25 units/μl) (all of the reagents were from Applied Biosystems) and incubated for 10 min at 25 °C followed by 30 min at 48 °C and 5 min at 95 °C. .. Real time PCR was performed using TaqMan system 7900HT (Applied Biosystems).

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  • 93
    Thermo Fisher purification gst mnase
    MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using <t>GST-RHΔ-MNase</t> (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p
    Purification Gst Mnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/purification gst mnase/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    purification gst mnase - by Bioz Stars, 2020-05
    93/100 stars
      Buy from Supplier

    Image Search Results


    MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using GST-RHΔ-MNase (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p

    Journal: Cell reports

    Article Title: Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach

    doi: 10.1016/j.celrep.2019.09.052

    Figure Lengend Snippet: MapR and RHΔ CUT RUN Signals Are Enriched at Similar Regions Genome-wide (A) Schematic of RHΔC R using FLAG M2 antibody (left) and MapR using GST-RHΔ-MNase (right) in HEK293. (B) Enriched regions identified by RHΔC R and R-ChIP in HEK293. GRO-seq and H3K4me3 tracks indicate active gene transcription. (C) Venn diagram of gene-level overlap between RHΔC R and R-ChIP. Total number of unique genes with an R-loop at the promoter region (−2kb/+2kb from the TSS) and their overlap are shown. p

    Article Snippet: Protein Expression and Purification GST-MNase and GST-RHΔMNase were cloned into pGEX plasmid and transformed into BL21 (DE3) (ThermoFisher C601003) for expression.

    Techniques: Genome Wide, Chromatin Immunoprecipitation

    MapR, a Native and Antibody-Independent R-Loop Detection Strategy R-loop recognition and recovery by MapR. Step 1: cells are immobilized on concanavalin A beads and permeabilized. Step 2: equimolar amounts of a catalytic deficient mutant of RNase H fused to micrococcal nuclease (GST-RHΔ-MNase) or GST-MNase is added to immobilized cells. Step 3: the RHΔ module recognizes and binds R-loops on chromatin. Step 4: controlled activation of the MNase moiety by addition of calcium results in cleavage of DNA fragments in proximity to R-loops. Step 5: Released R-loops diffuse out of the cell; the DNA is recovered and sequenced.

    Journal: Cell reports

    Article Title: Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach

    doi: 10.1016/j.celrep.2019.09.052

    Figure Lengend Snippet: MapR, a Native and Antibody-Independent R-Loop Detection Strategy R-loop recognition and recovery by MapR. Step 1: cells are immobilized on concanavalin A beads and permeabilized. Step 2: equimolar amounts of a catalytic deficient mutant of RNase H fused to micrococcal nuclease (GST-RHΔ-MNase) or GST-MNase is added to immobilized cells. Step 3: the RHΔ module recognizes and binds R-loops on chromatin. Step 4: controlled activation of the MNase moiety by addition of calcium results in cleavage of DNA fragments in proximity to R-loops. Step 5: Released R-loops diffuse out of the cell; the DNA is recovered and sequenced.

    Article Snippet: Protein Expression and Purification GST-MNase and GST-RHΔMNase were cloned into pGEX plasmid and transformed into BL21 (DE3) (ThermoFisher C601003) for expression.

    Techniques: Mutagenesis, Activation Assay