buffer 1  (New England Biolabs)


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    Name:
    Monarch Plasmid Wash Buffer 1
    Description:
    Monarch Plasmid Wash Buffer 1 54 ml
    Catalog Number:
    T1014L
    Price:
    34
    Size:
    54 ml
    Category:
    Plasmid Purification Kit Components
    Score:
    85
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    Structured Review

    New England Biolabs buffer 1
    Monarch Plasmid Wash Buffer 1
    Monarch Plasmid Wash Buffer 1 54 ml
    https://www.bioz.com/result/buffer 1/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    buffer 1 - by Bioz Stars, 2019-10
    99/100 stars

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    Related Articles

    Flow Cytometry:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n=2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Amplification:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min. .. 1 ng of purified total RNA, as determined by Agilent Bioanalyzer (Agilent Technologies), was processed with the mRNA direct micro kit (Life Technologies) to select for poly A RNA.

    In Vitro:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min. .. 1 ng of purified total RNA, as determined by Agilent Bioanalyzer (Agilent Technologies), was processed with the mRNA direct micro kit (Life Technologies) to select for poly A RNA.

    Isolation:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: After a series of washes in PBST (PBS and 0.1% Tween-20), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:1000 dilution) was added to the membrane and detected using ECL Western Blotting reagent (Amersham Biosciences). .. One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). .. The product DNA was separated on a 0.8% agarose gel.

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Paragraph title: RNA isolation ... The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Cytometry:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n=2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Mouse Assay:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n=2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Concentration Assay:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min. .. Each entire sample was input into the Ambion WT Expression Kit (Life Technologies) to perform double stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA.

    Incubation:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: After a series of washes in PBST (PBS and 0.1% Tween-20), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:1000 dilution) was added to the membrane and detected using ECL Western Blotting reagent (Amersham Biosciences). .. One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). .. The product DNA was separated on a 0.8% agarose gel.

    Activity Assay:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: Paragraph title: Biochemical R.HpyAXII endonuclease activity assay ... One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs).

    Expressing:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: After a series of washes in PBST (PBS and 0.1% Tween-20), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:1000 dilution) was added to the membrane and detected using ECL Western Blotting reagent (Amersham Biosciences). .. One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). .. The product DNA was separated on a 0.8% agarose gel.

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min. .. 1 ng of purified total RNA, as determined by Agilent Bioanalyzer (Agilent Technologies), was processed with the mRNA direct micro kit (Life Technologies) to select for poly A RNA.

    Lysis:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n=2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Injection:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: Cells were isolated by flow cytometry into RNEasy lysis buffer (Qiagen) from n=2–3 mice per condition, 1.5 days after injection of the appropriate adenoviruses. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Purification:

    Article Title: Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem cell self-renewal
    Article Snippet: The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min. .. The samples were then digested with 0.6 mAU Proteinase K (Qiagen) in the presence of 1× NEB Buffer 1 (NEB) at 50°C for 20 min, followed by a heat inactivation step at 65°C for 10 min. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen) at 37°C for 30 min.

    Plasmid Preparation:

    Article Title: The Helicobacter pylori HpyAXII restriction-modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components
    Article Snippet: After a series of washes in PBST (PBS and 0.1% Tween-20), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:1000 dilution) was added to the membrane and detected using ECL Western Blotting reagent (Amersham Biosciences). .. One microliter of freshly prepared soluble fraction obtained from E. coli BL21(DE3) expressing R.HpyAXII was incubated for 3 h at 37°C with 500 ng E. coli ER2925 genomic DNA or pET15b plasmid DNA isolated from E. coli DH10B in presence of NEB buffer 1 (New England Biolabs). .. The product DNA was separated on a 0.8% agarose gel.

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    New England Biolabs t4 dna ligase buffer
    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase buffer/product/New England Biolabs
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    82
    New England Biolabs l30 binding buffer
    REMSA analysis of the RNA-binding activity of <t>L30.</t> A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.
    L30 Binding Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs cas9 buffer
    Architecture of the SpyCas9-sgRNA in complex with AcrIIA4. ( A ) Cryo-EM reconstruction of the AcrII4-bound SpyCas9. The electron density map was contoured at high-threshold levels showing distinct features for each subunit. ( B ) Representative cryo-EM density for AcrIIA4 with the refined model superimposed. ( C ) The atomic model of SpyCas9-sgRNA-ArcrIIA4. AcrIIA4 (red) and sgRNA (orange) are shown in a ribbon diagram. ( D ) Surface representation showing AcrIIA4 binding to the PAM-recognition cleft. ( E ) Superposition with <t>Cas9–sgRNA–dsDNA</t> (double-stranded DNA) structure (PDB ID: 5F9R). For clarity, Cas9 is omitted except the HNH domain. Target and nontarget DNA strand is colored purple and beige, respectively. ( F ) Electrostatic surface potential of AcrIIA4 showing that AcrIIA4 fits perfectly into the major groove of PAM duplex and that its surface acts as a dsDNA mimic. The inset shows that the PAM recognition residues (R1333 and R1334) are largely buried in an acidic pocket within AcrIIA4.
    Cas9 Buffer, supplied by New England Biolabs, 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/cas9 buffer/product/New England Biolabs
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    Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat  = 2.2 ± 0.2 × 10 −4  s −1  and K M  = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0  was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat  is 8 × 10 −3  s −1  with an upper threshold for the K M  estimated to be 1 nM.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Estimation of Michaelis–Menten parameters for the ligation of DNA splinted by RNA for ( A ) T4 DNA ligase and ( B ) PBCV-1 DNA ligase. Reactions were carried out in ligase assay buffer with 1 mM ATP at 25°C. Initial reaction velocity for the consumption of substrate was measured through fits to the linear region of the reaction (generally the first ∼15% of reaction) with error bars taking into account the uncertainty in the linear fits and initial substrate and enzyme concentrations. Kinetic parameters were determined through fitting the Michaelis–Menten equation to the data by non-linear regression as described in Materials and Methods. For (A), in all reactions the only detected product was AppDNA, and the determined parameters for substrate consumption were k cat = 2.2 ± 0.2 × 10 −4 s −1 and K M = 300 ± 70 nM. For (B), the reaction products were a ∼ 3:1 mixture of ligated DNA to AppDNA, and the observed V 0 /[E] 0 was independent of substrate concentration over the range 0.5 nM–100 nM. The approximate k cat is 8 × 10 −3 s −1 with an upper threshold for the K M estimated to be 1 nM.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Concentration Assay

    Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of DNA splinted by RNA. (Left) Outline of the ligation assay: a 5′-phosphorylated, 3′-FAM labelled DNA ‘donor’ oligonucleotide and an unmodified DNA ‘acceptor’ oligonucleotide are annealed to a complementary RNA or DNA splint. This substrate was reacted with a ligase to form a mixture of unreacted starting material, adenylylated DNA and ligated product. The products were denatured, separated on CE, and detected by fluorescence. (Right) ligation of the standard RNA-splinted substrate in ligase assay buffer for 15 min at 25°C with ( A ) no enzyme, ( B ) 1 µM T4 DNA ligase and 10 µM ATP, ( C ) 1 µM T4 DNA ligase and 1 mM ATP, ( D ) 100 nM PBCV-1 DNA ligase and 10 µM ATP, and ( E ) 100 nM PBCV-1 DNA ligase and 1 mM ATP. Indicated peaks correspond to starting pDNA (I), AppDNA (II) and ligated product (III) as determined by coelution with synthetically prepared standards.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Fluorescence

    Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q  for each experiment was recorded, with lower C q  indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q  after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Detection of defined amounts of luciferase mRNA from a mixture with Jurkat total RNA through ligation of specific DNA probes (Probe set A) and detection by qPCR. RNA/DNA probe mixtures were annealed then incubated with either T4 DNA ligase (open circle) or PBCV-1 DNA ligase (×). The qPCR C q for each experiment was recorded, with lower C q indicating higher concentration of ligated probes. ( A ) Ligation time course with either 1 (solid line) or 0.01 ng (dotted line) of luciferase mRNA target, 0–8 h ligation time. ( B ) Dependence of C q after probe ligation with target luciferase mRNA present over a 7 log concentration range using a 2 h ligation time. NTC = no template control. The error bars show one standard deviation of the average of a minimum of three replicates.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Luciferase, Ligation, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay, Standard Deviation

    Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Journal: Nucleic Acids Research

    Article Title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase

    doi: 10.1093/nar/gkt1032

    Figure Lengend Snippet: Ligation of RNA-splinted DNA substrates by PBCV-1 and T4 DNA ligases under general reaction conditions. The 16 RNA-splinted DNA substrates, representing all possible base pairs at the ligation junction, were reacted and the extent of ligation and abortive adenylylation measured. The bases listed on the X axis (dN/pdN) refer to the identity of the base of the DNA acceptor at the ligation junction (dN) and the identity of the phosphorylated base on the donor at the ligation junction (pdN). For all substrates, the correct Watson–Crick base-pairing partner was present in the RNA splint. All reactions were incubated at 37°C in 50 mM Tris pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 100 nM RNA-splinted DNA substrate and ( A ) 100 nM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( B ) 100 nM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( C ) 1 µM PBCV-1 DNA ligase and 1 mM ATP for 15 min; ( D ) 1 µM PBCV-1 DNA ligase and 10 µM ATP for 15 min; ( E ) 1 µM T4 DNA ligase and 10 µM ATP for 15 min; and ( F ) 1 µM T4 DNA ligase and 10 µM ATP for 4 h. Here the total height of the bar indicates the fraction of starting material converted to products, with the solid portion indicating ligation product yield and the hashed portion indicating AppDNA yield.

    Article Snippet: T4 DNA ligase buffer (50 mM Tris pH 7.5 @ 25°C, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) was obtained as a 10× stock from New England BioLabs, Inc. (NEB, Ipswich, MA).

    Techniques: Ligation, Dominant Negative Mutation, Incubation

    REMSA analysis of the RNA-binding activity of L30. A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: REMSA analysis of the RNA-binding activity of L30. A . The 32 P-labeled PHGPx SECIS was incubated with increasing amounts of L30 as indicated. The complexes were analyzed by native gel electrophoresis (top panel). The percent SECIS bound at each protein concentration is shown graphically (bottom panel). B . REMSA analysis was performed as described in (A) except that the stem-loop from the L30 pre-mRNA (L30 RNA) was used as the probe.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Activity Assay, Labeling, Incubation, Nucleic Acid Electrophoresis, Protein Concentration

    L30 repression of UGA recoding correlates with SECIS-binding. UGA recoding assays were performed using the luc/UGA/TR1 reporter construct as described in the legend to Figure 4 A. Reactions contained either no L30 or 44 pmol of wild-type or mutant L30 proteins. Results are expressed as means ± SEM.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: L30 repression of UGA recoding correlates with SECIS-binding. UGA recoding assays were performed using the luc/UGA/TR1 reporter construct as described in the legend to Figure 4 A. Reactions contained either no L30 or 44 pmol of wild-type or mutant L30 proteins. Results are expressed as means ± SEM.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Binding Assay, Construct, Mutagenesis

    The U40C mutation abrogates L30 binding. A . Schematic illustrating the binding sites of SBP2 [ 14 ] and L30 (this work) on the SECIS, as determined by RNA footprinting. The position of the U40C point mutation is indicated. B . UV cross-linking experiments were performed using the 32 P-labeled wild-type PHGPx SECIS or the U40C mutant RNA, which were incubated with increasing amounts of purified L30 as indicated. After RNase digestion, the products were analyzed by SDS-PAGE and autoradiography.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: The U40C mutation abrogates L30 binding. A . Schematic illustrating the binding sites of SBP2 [ 14 ] and L30 (this work) on the SECIS, as determined by RNA footprinting. The position of the U40C point mutation is indicated. B . UV cross-linking experiments were performed using the 32 P-labeled wild-type PHGPx SECIS or the U40C mutant RNA, which were incubated with increasing amounts of purified L30 as indicated. After RNase digestion, the products were analyzed by SDS-PAGE and autoradiography.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Mutagenesis, Binding Assay, Footprinting, Labeling, Incubation, Purification, SDS Page, Autoradiography

    Repression of UGA recoding by L30 is rescued by SBP2. A . In vitro UGA recoding assays were performed using a luciferase reporter RNA that contains UGA (top panel) or UGU (bottom panel) at position 258 of the coding region, fused to either the PHGPx or TR1 SECIS element as indicated. Translation assays were performed in the presence of increasing amounts of L30 as indicated. The products were analyzed for luciferase activity using a luminometer, and the results are expressed as means ± SEM. Statistical significance is indicated by ** ( p

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Repression of UGA recoding by L30 is rescued by SBP2. A . In vitro UGA recoding assays were performed using a luciferase reporter RNA that contains UGA (top panel) or UGU (bottom panel) at position 258 of the coding region, fused to either the PHGPx or TR1 SECIS element as indicated. Translation assays were performed in the presence of increasing amounts of L30 as indicated. The products were analyzed for luciferase activity using a luminometer, and the results are expressed as means ± SEM. Statistical significance is indicated by ** ( p

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: In Vitro, Luciferase, Activity Assay

    Mutational analysis of L30. A . A schematic illustrating the primary sequences and predicted secondary structures of L30 and SBP2. The position numbers refer to the rat protein sequences. The L7Ae conserved RNA-binding domain is underlined and the conserved signature amino acid motifs for L30 and SBP2 are boxed as described in [ 37 ]. Arrows indicate the amino acids in L30 that were mutated to alanine. B . The wild-type and mutant L30 protein were expressed in bacteria, purified and analyzed by SDS-PAGE and Coomassie Blue staining. The molecular weight markers are shown in the left lane of each gel.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Mutational analysis of L30. A . A schematic illustrating the primary sequences and predicted secondary structures of L30 and SBP2. The position numbers refer to the rat protein sequences. The L7Ae conserved RNA-binding domain is underlined and the conserved signature amino acid motifs for L30 and SBP2 are boxed as described in [ 37 ]. Arrows indicate the amino acids in L30 that were mutated to alanine. B . The wild-type and mutant L30 protein were expressed in bacteria, purified and analyzed by SDS-PAGE and Coomassie Blue staining. The molecular weight markers are shown in the left lane of each gel.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Mutagenesis, Purification, SDS Page, Staining, Molecular Weight

    Point mutations in L30 affect RNA-binding activity. A . Representative REMSA analysis of the 32 P-labeled PHGPx SECIS (top panel) or L30 RNA (bottom panel). The RNA probes were incubated in the absence or presence of wild-type and mutant L30 proteins as indicated. The positions of the free probes and protein:RNA complexes are indicated. B . Graphical representation of REMSA results for L30 binding to the SECIS (top panel) or L30 RNA (bottom panel) from 5 or 3 independent experiments, respectively. The results are expressed relative to the activity of the wild-type protein, which is expressed as 100%. Statistical significance is shown by asterisks, with * (p

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: Point mutations in L30 affect RNA-binding activity. A . Representative REMSA analysis of the 32 P-labeled PHGPx SECIS (top panel) or L30 RNA (bottom panel). The RNA probes were incubated in the absence or presence of wild-type and mutant L30 proteins as indicated. The positions of the free probes and protein:RNA complexes are indicated. B . Graphical representation of REMSA results for L30 binding to the SECIS (top panel) or L30 RNA (bottom panel) from 5 or 3 independent experiments, respectively. The results are expressed relative to the activity of the wild-type protein, which is expressed as 100%. Statistical significance is shown by asterisks, with * (p

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: RNA Binding Assay, Activity Assay, Labeling, Incubation, Mutagenesis, Binding Assay

    RNA footprinting of the L30:SECIS complex. A . The structure of the SECIS element from the rat PHGPx mRNA is shown. Boxes and circles indicate nucleotides that are protected from cleavage by RNase T1 and RNase A, respectively. B . The 5’ end-labeled PHGPx SECIS was incubated in the absence or presence of L30 (0.75 or 1.5 μM). The reactions were then partially digested with RNase T1, A, or V1 as indicated. The products were analyzed by denaturing gel electrophoresis. The sequencing (G and C + U) and alkali ladders are shown in the left lanes. The numbers to the left of the gel indicate the positions of G nucleotides using the numbering in (A). The bars on the right indicate the different regions of the SECIS element. The gel is a representative example from 3 independent experiments.

    Journal: BMC Molecular Biology

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element

    doi: 10.1186/1471-2199-14-12

    Figure Lengend Snippet: RNA footprinting of the L30:SECIS complex. A . The structure of the SECIS element from the rat PHGPx mRNA is shown. Boxes and circles indicate nucleotides that are protected from cleavage by RNase T1 and RNase A, respectively. B . The 5’ end-labeled PHGPx SECIS was incubated in the absence or presence of L30 (0.75 or 1.5 μM). The reactions were then partially digested with RNase T1, A, or V1 as indicated. The products were analyzed by denaturing gel electrophoresis. The sequencing (G and C + U) and alkali ladders are shown in the left lanes. The numbers to the left of the gel indicate the positions of G nucleotides using the numbering in (A). The bars on the right indicate the different regions of the SECIS element. The gel is a representative example from 3 independent experiments.

    Article Snippet: The RNA (2.5 nM) was incubated in L30 binding buffer (30 mM Tris HCl, pH 8.0, 75 mM KCl, 5 mM MgCl2 , 1 mM DTT, 0.04 μg/μL BSA (NEB), 10% glycerol, and 50 ng/μL yeast tRNA) with or without rat L30 protein (0.75 or 1.5 μM as indicated) at 30°C for 15 min.

    Techniques: Footprinting, Labeling, Incubation, Nucleic Acid Electrophoresis, Sequencing

    Architecture of the SpyCas9-sgRNA in complex with AcrIIA4. ( A ) Cryo-EM reconstruction of the AcrII4-bound SpyCas9. The electron density map was contoured at high-threshold levels showing distinct features for each subunit. ( B ) Representative cryo-EM density for AcrIIA4 with the refined model superimposed. ( C ) The atomic model of SpyCas9-sgRNA-ArcrIIA4. AcrIIA4 (red) and sgRNA (orange) are shown in a ribbon diagram. ( D ) Surface representation showing AcrIIA4 binding to the PAM-recognition cleft. ( E ) Superposition with Cas9–sgRNA–dsDNA (double-stranded DNA) structure (PDB ID: 5F9R). For clarity, Cas9 is omitted except the HNH domain. Target and nontarget DNA strand is colored purple and beige, respectively. ( F ) Electrostatic surface potential of AcrIIA4 showing that AcrIIA4 fits perfectly into the major groove of PAM duplex and that its surface acts as a dsDNA mimic. The inset shows that the PAM recognition residues (R1333 and R1334) are largely buried in an acidic pocket within AcrIIA4.

    Journal: Science Advances

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic

    doi: 10.1126/sciadv.1701620

    Figure Lengend Snippet: Architecture of the SpyCas9-sgRNA in complex with AcrIIA4. ( A ) Cryo-EM reconstruction of the AcrII4-bound SpyCas9. The electron density map was contoured at high-threshold levels showing distinct features for each subunit. ( B ) Representative cryo-EM density for AcrIIA4 with the refined model superimposed. ( C ) The atomic model of SpyCas9-sgRNA-ArcrIIA4. AcrIIA4 (red) and sgRNA (orange) are shown in a ribbon diagram. ( D ) Surface representation showing AcrIIA4 binding to the PAM-recognition cleft. ( E ) Superposition with Cas9–sgRNA–dsDNA (double-stranded DNA) structure (PDB ID: 5F9R). For clarity, Cas9 is omitted except the HNH domain. Target and nontarget DNA strand is colored purple and beige, respectively. ( F ) Electrostatic surface potential of AcrIIA4 showing that AcrIIA4 fits perfectly into the major groove of PAM duplex and that its surface acts as a dsDNA mimic. The inset shows that the PAM recognition residues (R1333 and R1334) are largely buried in an acidic pocket within AcrIIA4.

    Article Snippet: Labeled and unlabeled oligonucleotides were annealed in the presence of NEB Cas9 buffer [20 mM Hepes, 100 mM NaCl, 5 mM MgCl2 , and 0.1 mM EDTA (pH 6.5)] after a 2-min heat denaturation at 95°C.

    Techniques: Electron Microscopy, Binding Assay

    AcrIIA4 binds to the SpyCas9-sgRNA complex. ( A ) A cartoon depiction of Cas9 protein loaded with the sgRNA binding to AcrIIA4 (pink). Cas9-sgRNA complexed with AcrIIA4 is unable to bind to the target DNA. ( B ) Size exclusion chromatogram of SpyCas9-sgRNA in the presence or absence of sgRNA after preincubation with AcrIIA4. Relevant peaks are indicated with arrowheads. ( C ) Coomassie (CCB)– and ethidium bromide (EB)–stained polyacrylamide gel showing the comigration of AcrIIA4 with Cas9 in the presence of gRNA.

    Journal: Science Advances

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic

    doi: 10.1126/sciadv.1701620

    Figure Lengend Snippet: AcrIIA4 binds to the SpyCas9-sgRNA complex. ( A ) A cartoon depiction of Cas9 protein loaded with the sgRNA binding to AcrIIA4 (pink). Cas9-sgRNA complexed with AcrIIA4 is unable to bind to the target DNA. ( B ) Size exclusion chromatogram of SpyCas9-sgRNA in the presence or absence of sgRNA after preincubation with AcrIIA4. Relevant peaks are indicated with arrowheads. ( C ) Coomassie (CCB)– and ethidium bromide (EB)–stained polyacrylamide gel showing the comigration of AcrIIA4 with Cas9 in the presence of gRNA.

    Article Snippet: Labeled and unlabeled oligonucleotides were annealed in the presence of NEB Cas9 buffer [20 mM Hepes, 100 mM NaCl, 5 mM MgCl2 , and 0.1 mM EDTA (pH 6.5)] after a 2-min heat denaturation at 95°C.

    Techniques: Binding Assay, Staining

    Timed delivery of AcrIIA4 differentially inhibits on- and off-target genome editing in human cells. ( A ) Simultaneous delivery of Cas9 RNP and AcrIIA4 inhibits Cas9-mediated gene targeting in human cells. K562 cells with a chromosomally integrated BFP (BFP-K562) were nucleofected with Cas9 RNP and AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:0.5, 1:1, 1:2, 1:3, and 1:5] or plasmid (0.7 and 2.8 μg). Nonhomologous end joining (NHEJ) frequencies are quantified by the loss of BFP expression in BFP-K562 cells 96 hours after nucleofection via flow cytometry. Data are presented as means ± SEM from at least two biological replicates. ( B ) Administration of AcrIIA4 before Cas9 RNP completely inhibits Cas9-mediated gene targeting. BFP-K562 cells were nucleofected with AcrIIA4 plasmid (0.7 μg) 24 hours before Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. ( C ) Delivery of AcrIIA4 after introduction of Cas9 RNP yields intermediate inhibition of Cas9 activity. BFP-K562 cells were nucleofected with AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) 6 hours after Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. ( D ) Proper timing of AcrIIA4 delivery diminishes off-target editing events while largely retaining on-target editing. K562 cells were nucleofected with either HBB or EMX1 targeting Cas9 RNP 6 hours before AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) delivery ( 18 ). Representative T7 endonuclease I assay for visualization of HBB on- and off-target editing. Bottom: Inhibition of editing by AcrIIA4 at on- and off-target sites of HBB and VEGFA, as measured by amplicon sequencing.

    Journal: Science Advances

    Article Title: Disabling Cas9 by an anti-CRISPR DNA mimic

    doi: 10.1126/sciadv.1701620

    Figure Lengend Snippet: Timed delivery of AcrIIA4 differentially inhibits on- and off-target genome editing in human cells. ( A ) Simultaneous delivery of Cas9 RNP and AcrIIA4 inhibits Cas9-mediated gene targeting in human cells. K562 cells with a chromosomally integrated BFP (BFP-K562) were nucleofected with Cas9 RNP and AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:0.5, 1:1, 1:2, 1:3, and 1:5] or plasmid (0.7 and 2.8 μg). Nonhomologous end joining (NHEJ) frequencies are quantified by the loss of BFP expression in BFP-K562 cells 96 hours after nucleofection via flow cytometry. Data are presented as means ± SEM from at least two biological replicates. ( B ) Administration of AcrIIA4 before Cas9 RNP completely inhibits Cas9-mediated gene targeting. BFP-K562 cells were nucleofected with AcrIIA4 plasmid (0.7 μg) 24 hours before Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. ( C ) Delivery of AcrIIA4 after introduction of Cas9 RNP yields intermediate inhibition of Cas9 activity. BFP-K562 cells were nucleofected with AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) 6 hours after Cas9 RNP delivery. Data are presented as means ± SEM from at least two biological replicates. ( D ) Proper timing of AcrIIA4 delivery diminishes off-target editing events while largely retaining on-target editing. K562 cells were nucleofected with either HBB or EMX1 targeting Cas9 RNP 6 hours before AcrIIA4 protein [Cas9/AcrIIA4 (molar ratio), 1:5] or plasmid (0.7 μg) delivery ( 18 ). Representative T7 endonuclease I assay for visualization of HBB on- and off-target editing. Bottom: Inhibition of editing by AcrIIA4 at on- and off-target sites of HBB and VEGFA, as measured by amplicon sequencing.

    Article Snippet: Labeled and unlabeled oligonucleotides were annealed in the presence of NEB Cas9 buffer [20 mM Hepes, 100 mM NaCl, 5 mM MgCl2 , and 0.1 mM EDTA (pH 6.5)] after a 2-min heat denaturation at 95°C.

    Techniques: Plasmid Preparation, Non-Homologous End Joining, Expressing, Flow Cytometry, Cytometry, Inhibition, Activity Assay, T7EI Assay, Amplification, Sequencing