rnase inhibitor  (New England Biolabs)


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    Name:
    RNase Inhibitor Murine
    Description:
    RNase Inhibitor Murine 15 000 units
    Catalog Number:
    m0314l
    Price:
    280
    Size:
    15 000 units
    Category:
    Enzyme Inhibitors
    Buy from Supplier


    Structured Review

    New England Biolabs rnase inhibitor
    RNase Inhibitor Murine
    RNase Inhibitor Murine 15 000 units
    https://www.bioz.com/result/rnase inhibitor/product/New England Biolabs
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-01
    90/100 stars

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    Related Articles

    Clone Assay:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Mitochondrial transcription termination factor 1 directs polar replication fork pausing
    Article Snippet: In vitro transcription experiments The HSP and LSP transcription templates were cloned as described in ( ) and linearized with PstI and EcoRI respectively. .. The transcription reaction volumes were 25 μl and contained 25 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 40 mM NaCl, 100 μg/mL BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 4 nM of indicated plasmid template.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end (for a C-terminal tag) and cloned into the pJS119K vector ( ).

    Centrifugation:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
    Article Snippet: RNA-immunoprecipitation 50 ODs of cells were grown at 30°C in EMM 0.5X and harvested by centrifugation. .. Cell pellets were resuspended in 2 ml lysis buffer (6 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaC2 H3 O2 , 5 mM MgC2 H3 O2 , 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM AEBSF, 4 mM benzamidine, 2X Roche complete EDTA-free protease inhibitor cocktail and 160 U Murine RNase inhibitor (New England Biolabs, #M0314L)) to make ‘pop-corn’.

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]). .. The supernatant was removed, and the nuclei were washed with 1 mL of lysis buffer B followed by centrifugation at 1000 g for 3 min.

    Amplification:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Autoradiography:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: Paragraph title: 35 S metabolic labeling, immunoprecipitation, and autoradiography ... The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling.

    Construct:

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Most transcription reactions were carried out with constructs in which the nontemplate strand extends only to position +2, a common practice in the field ( , ). .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Real-time Polymerase Chain Reaction:

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    Incubation:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. In parallel, the antibody against the protein of interest was incubated with protein G–Sepharose beads on a head-over-tail rotor for 1 h at RT and washed once with 1× PBS and twice with lysis buffer.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs). .. Incubation with calcein AM (100 nM, room temperature, 30 min) and then with CD45 and CD31 antibodies (catalogs 103114 and 102508, BioLegend) on ice (30 min) was followed by 7-AAD (5 μg/ml) for an additional 10 minutes before the end of the incubation.

    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
    Article Snippet: .. After two washes with PBS, the anti-Flag-coated beads were added to the poly(A) selected RNA extract in 0.5 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton, and 1 µl Murine RNAse inhibitor (NEB-M0314S)) and incubated for 1.5 h in 4 °C. .. Precleared RNA samples were then subjected to 5′-CAP RNA immunoprecipitation using 5 µl (1:100 dilution) anti-Cap antibody (Merck anti-m3G-cap, m7G-cap antibody, clone H-20) and incubated O/N in 4 °C.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations. .. The reactions were stopped after 30 min at 32°C by the addition of stop buffer (10 mM Tris–HCl, pH 8.0, 0.2 M NaCl, 1 mM EDTA and 100 μg/ml proteinase K) followed by incubation at 42°C for 45 min.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The reaction was incubated at 37°C for 4 h. Primer extension on synthetic 24-mer RNA (purchased from IDT, sequence is shown in ) was performed under the above ‘low yield’ condition, with 25 μM RNA replacing template DNA. .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Activity Assay:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity. ..

    Expressing:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: Paragraph title: In vitro and in vivo sialidase expression ... For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Cell Fractionation:

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: Cellular fractionation was performed as described previously ( ) with minor modifications. .. Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]).

    Gel Purification:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Protease Inhibitor:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
    Article Snippet: .. Cell pellets were resuspended in 2 ml lysis buffer (6 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaC2 H3 O2 , 5 mM MgC2 H3 O2 , 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM AEBSF, 4 mM benzamidine, 2X Roche complete EDTA-free protease inhibitor cocktail and 160 U Murine RNase inhibitor (New England Biolabs, #M0314L)) to make ‘pop-corn’. ..

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Cell Culture:

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    Generated:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: PURExpress DNA templates were generated by PCR using primers specific to ORF9 or ORF12 ( Table S1 ). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    other:

    Article Title: Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification
    Article Snippet: Transcriptions were done in 1× transcription buffer (40 mM Tris, 10 mM dithiothreitol, 2 mM spermidine, 0.002% Triton X-100, and 27 mM magnesium acetate) using final concentrations of 8 U/μL T7 RNA polymerase (M0251L); 0.002 U/μL inorganic pyrophosphatase (M2403L); 1 U/μL murine RNase inhibitor (M0314L); 0.025 μg/μL standard or uridine-depleted transcription template; 5 mM CleanCap Cap 1 AG trimer; and 5 mM each of ATP, cytidine triphosphate (CTP) (or CTP analog), GTP, and uridine triphosphate (UTP) (or UTP analog), as indicated in .

    Polymerase Chain Reaction:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Binding Assay:

    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
    Article Snippet: .. After two washes with PBS, the anti-Flag-coated beads were added to the poly(A) selected RNA extract in 0.5 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton, and 1 µl Murine RNAse inhibitor (NEB-M0314S)) and incubated for 1.5 h in 4 °C. .. Precleared RNA samples were then subjected to 5′-CAP RNA immunoprecipitation using 5 µl (1:100 dilution) anti-Cap antibody (Merck anti-m3G-cap, m7G-cap antibody, clone H-20) and incubated O/N in 4 °C.

    In Vivo:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: Paragraph title: In vitro and in vivo sialidase expression ... For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Fluorescence:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Additionally, sialidase activity of in vitro expressed proteins was assessed by incubating 20 μl of PURExpress product with 4 μl of 100 μg/ml 4MU-α-Neu5Ac at 37 °C for 1 h and reading fluorescence at λex = 365 nm and λem = 445 nm in a SpectraMax microplate fluorometer.

    Isolation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: CMs were pelleted and subjected to Percoll density gradient centrifugation (1,200 g for 30 min) for RNA and protein isolation. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Labeling:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Purification:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations. .. The transcripts were purified with ethanol precipitation and the pellets were dissolved in 20 μl gel loading buffer (98% formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue) and heated at 95°C for 5 min.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: The amplified product was purified using the Monarch PCR clean-up kit (New England Biolabs). .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein.

    Sequencing:

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end (for a C-terminal tag) and cloned into the pJS119K vector ( ).

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: The reaction was incubated at 37°C for 4 h. Primer extension on synthetic 24-mer RNA (purchased from IDT, sequence is shown in ) was performed under the above ‘low yield’ condition, with 25 μM RNA replacing template DNA. .. RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    FACS:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: Non-CMs in the supernatant were centrifuged at 500 g (10 min) and prepared for FACS. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Lysis:

    Article Title: Ubiquitination-dependent control of sexual differentiation in fission yeast
    Article Snippet: .. Cell pellets were resuspended in 2 ml lysis buffer (6 mM Na2 HPO4 , 4 mM NaH2 PO4 , 150 mM NaC2 H3 O2 , 5 mM MgC2 H3 O2 , 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM AEBSF, 4 mM benzamidine, 2X Roche complete EDTA-free protease inhibitor cocktail and 160 U Murine RNase inhibitor (New England Biolabs, #M0314L)) to make ‘pop-corn’. ..

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: .. The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling. .. Lysates were precleared using Sepharose–protein G beads (GE Healthcare) for 1 h at 4°C on a head-over-tail rotor.

    Article Title: Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP) *
    Article Snippet: .. We obtained yeast extract, peptone, and agar from BD Biosciences; S1P inhibitor PF-429242 from Shanghai APIs Chemical Co.; proteasome inhibitor MG132 (C2211), lysosome inhibitor ammonium chloride (A9434), mevalonolactone (M4667, for sodium mevalonate preparation), puromycin dihydrochloride (P8833), oleic acid-albumin (O3008), doxycycline (D9891), crystal violet (C3886), soybean trypsin inhibitor (T9003), glass beads (G8772, for yeast cell lysis), trypsin (T8003), and lipoprotein-deficient serum (LPDS; S5394) from Sigma-Aldrich (catalogue numbers in parentheses); cell culture media DMEM (10-013), DMEM/F12 (10-092), and penicillin-streptomycin (30-002) from Corning Cellgro; FuGENE 6 and RNase-free DNase I (10104159001) from Roche Applied Science; random primer mix (S1330), M-MuLV reverse transcriptase (M0253L), murine RNase inhibitor (M0314L), oligo d(T)23 VN (S1327S), and endoglycosidase Hf (P0703) from New England Biolabs; GoTaq real-time PCR mix (A6002) from Promega; SCAP trafficking inhibitor fatostatin (341329) and compactin (mevastatin, 474705) from Millipore; and BioCoatTM collagen-coated culture dish (VWR 62405-617) from BD Biosciences. .. We obtained wild-type haploid S. pombe KGY425 from ATCC.

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: .. Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]). ..

    Plasmid Preparation:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Linearization of the vector was performed by PCR with Q5 hot start high-fidelity 2× Master Mix (New England Biolabs) for 25 cycles (98 °C for 10 s, 60 °C for 30 s, and 72 °C for 2 min).

    Article Title: Mitochondrial transcription termination factor 1 directs polar replication fork pausing
    Article Snippet: .. The transcription reaction volumes were 25 μl and contained 25 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 40 mM NaCl, 100 μg/mL BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 4 nM of indicated plasmid template. ..

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. ORF12 was fused with a hexahistidine tag coding sequence at its 3′ end (for a C-terminal tag) and cloned into the pJS119K vector ( ).

    In Vitro:

    Article Title: POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA
    Article Snippet: .. In vitro transcription assay Transcription reactions were in 25 μl total volume containing 25 mM tris-HCl (pH 8.0), 10 mM MgCl2 , 64 mM NaCl, BSA (100 μg/ml), 1 mM dithiothreitol (DTT), 400 μM ATP, 150 μM guanosine-5′-triphosphate (GTP), 150 μM cytidine-5′-triphosphate (CTP), 10 μM uridine 5′-triphosphate (UTP), 0.02 μM [α-32 P]UTP, and 4 U RNase Inhibitor Murine (New England Biolabs). .. The transcription template was added at 4 nM and consisted of a restriction cut (Hind III/Eco RI), purified (QIAquick PCR Purification Kit) human LSP/HSP plasmid, where a fragment consisting of positions 325 to 742 of human mtDNA was cloned between the Sma I and Hind III sites of pUC18 ( ).

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: Mitochondrial transcription termination factor 1 directs polar replication fork pausing
    Article Snippet: Paragraph title: In vitro transcription experiments ... The transcription reaction volumes were 25 μl and contained 25 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 40 mM NaCl, 100 μg/mL BSA, 10 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 4 nM of indicated plasmid template.

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: Paragraph title: In vitro transcription ... All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)
    Article Snippet: .. For in vitro protein synthesis, 1 μl of amplified ORF9 or ORF12 DNA was mixed with 10 μl of solution A, 7.5 μl of solution B, and 0.5 μl of RNase inhibitor murine (New England Biolabs) and incubated for 2 h at 37 °C to express the desired protein. .. Expression was verified by separating 2.5 μl of the reaction on a Novex 10–20% Tris–glycine gel (Thermo Fisher Scientific).

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses
    Article Snippet: Paragraph title: In vitro transcription by T7 RNA polymerase ... RNase Inhibitor Murine (New England BioLabs) was added to each transcription mixture before and after the reaction, to inhibit any RNase activity.

    Ethanol Precipitation:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations. .. The transcripts were purified with ethanol precipitation and the pellets were dissolved in 20 μl gel loading buffer (98% formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromophenol blue) and heated at 95°C for 5 min.

    Produced:

    Article Title: The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation
    Article Snippet: The non-promoter template was produced by digestion of the LSP template with EcoRI followed by gel purification (Quiagen) of the vector fragment. .. All transcription reaction volumes were 25 μl and contained 25 mM Tris–HCl, pH 8.0, 10 mM MgCl2 , 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP 10 μM UTP, 0,02 μM α-32 P UTP (3000 Ci/mmol), 4 U RNase inhibitor Murine (New England Biolabs), 90 fmole of indicated template and proteins at indicated concentrations.

    Immunoprecipitation:

    Article Title: Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation
    Article Snippet: Paragraph title: 35 S metabolic labeling, immunoprecipitation, and autoradiography ... The cells were then washed extensively using 1× PBS and lysed in lysis buffer (50 mM Hepes-NaOH, pH 7.5, 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2 , 0.8 U/µl murine RNase inhibitor [M0314S; New England Biolabs, Inc.], and protease inhibitor cocktail) as described in pSILAC labeling.

    Article Title: Post-transcriptional 3´-UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments
    Article Snippet: Paragraph title: 5′- CAP RNA immunoprecipitation ... After two washes with PBS, the anti-Flag-coated beads were added to the poly(A) selected RNA extract in 0.5 ml binding buffer (50 mM Tris, 150 mM NaCl, 0.5% Triton, and 1 µl Murine RNAse inhibitor (NEB-M0314S)) and incubated for 1.5 h in 4 °C.

    Fractionation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: Paragraph title: Heart suborgan fractionation. ... RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

    Article Title: A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs
    Article Snippet: Paragraph title: Nuclear and cytoplasmic fractionation ... Briefly, cells were washed twice with serum-free medium (DL1 cells) or PBS (HeLa cells) and resuspended with slow pipetting in 1 mL of lysis buffer B (10 mM Tris-HCl at pH 8, 140 mM NaCl, 1.5 mM MgCl2 , 0.5% IGEPAL CA-630, 1 mM dithiothreitol, 80 U/mL RNase inhibitor [New England Biolabs, M0314L]).

    Staining:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs). .. Incubation with calcein AM (100 nM, room temperature, 30 min) and then with CD45 and CD31 antibodies (catalogs 103114 and 102508, BioLegend) on ice (30 min) was followed by 7-AAD (5 μg/ml) for an additional 10 minutes before the end of the incubation.

    Gradient Centrifugation:

    Article Title: Endothelial cell CD36 optimizes tissue fatty acid uptake
    Article Snippet: CMs were pelleted and subjected to Percoll density gradient centrifugation (1,200 g for 30 min) for RNA and protein isolation. .. RBC in non-CMs were lysed and pellets resuspended in staining buffer (2% FBS/PBS, 2 mM EDTA, and 100 U/ml RNase inhibitor; catalog M0314L, New England BioLabs).

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