low range ssrna ladder  (New England Biolabs)


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    Name:
    Low Range ssRNA Ladder
    Description:
    Low Range ssRNA Ladder 25 gel lanes
    Catalog Number:
    N0364S
    Price:
    69
    Category:
    RNA Ladders
    Size:
    100 gel lanes
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    Structured Review

    New England Biolabs low range ssrna ladder
    Low Range ssRNA Ladder
    Low Range ssRNA Ladder 25 gel lanes
    https://www.bioz.com/result/low range ssrna ladder/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    low range ssrna ladder - by Bioz Stars, 2021-05
    95/100 stars

    Images

    1) Product Images from "Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs"

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw786

    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).
    Figure Legend Snippet: Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Techniques Used: Incubation, In Vitro, In Vivo, Polyacrylamide Gel Electrophoresis, Staining, Purification, Negative Control

    2) Product Images from "Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A"

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

    Journal: bioRxiv

    doi: 10.1101/2021.01.31.429007

    Ribonuclease activity in the presence of EDTA and divalent cations. A. MBP-RNase I ribonuclease activity assay. The low range ssRNA ladder (50 to 1000 nt long, NEB) was used as the substrate for RNase activity assay in a high sale buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) supplemented with divalent cations (1 mM) or EDTA (10 mM).
    Figure Legend Snippet: Ribonuclease activity in the presence of EDTA and divalent cations. A. MBP-RNase I ribonuclease activity assay. The low range ssRNA ladder (50 to 1000 nt long, NEB) was used as the substrate for RNase activity assay in a high sale buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) supplemented with divalent cations (1 mM) or EDTA (10 mM).

    Techniques Used: Activity Assay

    3) Product Images from "In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex"

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku120

    Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.
    Figure Legend Snippet: Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Techniques Used: RNA Binding Assay, Recombinant, Incubation, Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Binding Assay, Labeling, Purification, Construct, Clear Native PAGE

    Related Articles

    Polyacrylamide Gel Electrophoresis:

    Article Title: Distinct E-cadherin-based complexes regulate cell behaviour through miRNA processing or Src and p120 catenin activity
    Article Snippet: The raw units were then normalized for RNA input and background noise to internal positive and negative controls according to the manufacturer’s instructions. miRNAs with low counts at the level of the negative controls were excluded. .. 10–20 μg of total RNA or the Low Range ssRNA Ladder (NEB) were mixed in a 1:1 volume ratio with Gel Loading Buffer II (Life Technologies), denatured at 95 °C for 5 min and loaded onto 12% denaturing PAGE RNA gels (SequaGel; National Diagnostics). .. RNAs were then transferred to Hybond N+ membranes (Amersham, GE Healthcare) for 2 h at 250 mA using a semidry apparatus (Hoefer TE70) and cross linked using a Stratalinker (Stratagene).

    Activity Assay:

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A
    Article Snippet: Previous studies showed that E. coli RNase I does not require divalent cations for nuclease activity, and the other eight ribonucleases from E. coli are only active in the presence of divalent cations [ ]. .. To demonstrate the ribonuclease activity for the partially purified MBP-RNase I, we incubated a low range ssRNA ladder (N0364S, NEB) with the enzyme (3 enzyme dilutions at 2 μg, 0.2 μg, and 0.02 μg) in a high salt buffer supplemented with EDTA, Ni2+ , Ca2+ , Mn2+ , Co2+ , or Mg2+ . ..

    Purification:

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A
    Article Snippet: Previous studies showed that E. coli RNase I does not require divalent cations for nuclease activity, and the other eight ribonucleases from E. coli are only active in the presence of divalent cations [ ]. .. To demonstrate the ribonuclease activity for the partially purified MBP-RNase I, we incubated a low range ssRNA ladder (N0364S, NEB) with the enzyme (3 enzyme dilutions at 2 μg, 0.2 μg, and 0.02 μg) in a high salt buffer supplemented with EDTA, Ni2+ , Ca2+ , Mn2+ , Co2+ , or Mg2+ . ..

    Article Title: Dynamic and scalable DNA-based information storage
    Article Snippet: RNA samples were treated with 2 units DNase I (NEB, M0303S) and incubated at 37 °C for 10 min, followed by a purification process using Monarch RNA Cleanup Kit (NEB, T2030S). .. The purified samples and RNA ladder (NEB, N0364S) were mixed with 1x RNA loading dye containing 47.5% Formamide, 0.01% SDS, 0.01% bromophenol blue, 0.005% Xylene Cyanol and 0.5 mM EDTA (NEB, B0363S). .. The mixtures were heated up at 65 C for 10 min, followed by immediate cooling on ice for 5 min. RNA electrophoresis was performed at a voltage gradient of 15 V/cm for 45 min.

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex
    Article Snippet: The crRNA was prepared by run-off transcription in a reaction containing 40 mM HEPES/KOH, pH 8.0, 22 mM MgCl2 , 5 mM dithiothreitol, 1 mM spermidine, 4 mM of ATP, CTP, GTP and UTP, 20 U RNase inhibitor, 1 µg T7 RNA polymerase and template DNA [PCR products with sequence-specific primers (cr5.2PCRf/r or cr5.13PCRf/r, respectively)] at 37°C for 4 h. For the self-cleaving reaction of the hammerhead ribozyme, the transcription reaction was directly diluted with 4 volumes of 30 mM MgCl2 in DEPC-H2 O and incubated for 1 h at 60°C. .. The cleaved crRNA was purified by phenol/chloroform extraction (pH 5.2), EtOH precipitated with the addition of glycogen (1:100, v/v), mixed with 2× formamide loading buffer (95% formamide, 5 mM EDTA, pH 8.0, 2.5 mg bromophenol blue, 2.5 mg xylene cyanol), heated for 5 min at 95°C, separated by a denaturing-PAGE (8 M urea, 1× TBE, 10% polyacrylamide) next to an RNA marker (low range ssRNA ladder, NEB) and visualized by toluidine blue staining. ..

    Incubation:

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A
    Article Snippet: Previous studies showed that E. coli RNase I does not require divalent cations for nuclease activity, and the other eight ribonucleases from E. coli are only active in the presence of divalent cations [ ]. .. To demonstrate the ribonuclease activity for the partially purified MBP-RNase I, we incubated a low range ssRNA ladder (N0364S, NEB) with the enzyme (3 enzyme dilutions at 2 μg, 0.2 μg, and 0.02 μg) in a high salt buffer supplemented with EDTA, Ni2+ , Ca2+ , Mn2+ , Co2+ , or Mg2+ . ..

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs
    Article Snippet: .. Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ). .. RNA was visualized after ethidium bromide staining under UV light (254 nm) in a gel documentation system (E-Box-3026, peQlab).

    Staining:

    Article Title: RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
    Article Snippet: To detect low molecular weight RNAs, RNA was analyzed by gel electrophoresis in 6% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA buffer. .. The ladders used were either a low-range ssRNA ladder (NEB), which was excised from the gel prior to transfer, stained with ethidium bromide for visualization with UV light and then realigned with the Northern blot, or the Decade Marker System (Ambion) which was Northern blotted directly. .. Northern Blots were analyzed by autoradiography and phosphorimager analysis.

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex
    Article Snippet: The crRNA was prepared by run-off transcription in a reaction containing 40 mM HEPES/KOH, pH 8.0, 22 mM MgCl2 , 5 mM dithiothreitol, 1 mM spermidine, 4 mM of ATP, CTP, GTP and UTP, 20 U RNase inhibitor, 1 µg T7 RNA polymerase and template DNA [PCR products with sequence-specific primers (cr5.2PCRf/r or cr5.13PCRf/r, respectively)] at 37°C for 4 h. For the self-cleaving reaction of the hammerhead ribozyme, the transcription reaction was directly diluted with 4 volumes of 30 mM MgCl2 in DEPC-H2 O and incubated for 1 h at 60°C. .. The cleaved crRNA was purified by phenol/chloroform extraction (pH 5.2), EtOH precipitated with the addition of glycogen (1:100, v/v), mixed with 2× formamide loading buffer (95% formamide, 5 mM EDTA, pH 8.0, 2.5 mg bromophenol blue, 2.5 mg xylene cyanol), heated for 5 min at 95°C, separated by a denaturing-PAGE (8 M urea, 1× TBE, 10% polyacrylamide) next to an RNA marker (low range ssRNA ladder, NEB) and visualized by toluidine blue staining. ..

    Northern Blot:

    Article Title: RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
    Article Snippet: To detect low molecular weight RNAs, RNA was analyzed by gel electrophoresis in 6% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA buffer. .. The ladders used were either a low-range ssRNA ladder (NEB), which was excised from the gel prior to transfer, stained with ethidium bromide for visualization with UV light and then realigned with the Northern blot, or the Decade Marker System (Ambion) which was Northern blotted directly. .. Northern Blots were analyzed by autoradiography and phosphorimager analysis.

    Marker:

    Article Title: RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V
    Article Snippet: To detect low molecular weight RNAs, RNA was analyzed by gel electrophoresis in 6% polyacrylamide gels containing 7 M urea in Tris-borate-EDTA buffer. .. The ladders used were either a low-range ssRNA ladder (NEB), which was excised from the gel prior to transfer, stained with ethidium bromide for visualization with UV light and then realigned with the Northern blot, or the Decade Marker System (Ambion) which was Northern blotted directly. .. Northern Blots were analyzed by autoradiography and phosphorimager analysis.

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex
    Article Snippet: The crRNA was prepared by run-off transcription in a reaction containing 40 mM HEPES/KOH, pH 8.0, 22 mM MgCl2 , 5 mM dithiothreitol, 1 mM spermidine, 4 mM of ATP, CTP, GTP and UTP, 20 U RNase inhibitor, 1 µg T7 RNA polymerase and template DNA [PCR products with sequence-specific primers (cr5.2PCRf/r or cr5.13PCRf/r, respectively)] at 37°C for 4 h. For the self-cleaving reaction of the hammerhead ribozyme, the transcription reaction was directly diluted with 4 volumes of 30 mM MgCl2 in DEPC-H2 O and incubated for 1 h at 60°C. .. The cleaved crRNA was purified by phenol/chloroform extraction (pH 5.2), EtOH precipitated with the addition of glycogen (1:100, v/v), mixed with 2× formamide loading buffer (95% formamide, 5 mM EDTA, pH 8.0, 2.5 mg bromophenol blue, 2.5 mg xylene cyanol), heated for 5 min at 95°C, separated by a denaturing-PAGE (8 M urea, 1× TBE, 10% polyacrylamide) next to an RNA marker (low range ssRNA ladder, NEB) and visualized by toluidine blue staining. ..

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs
    Article Snippet: .. Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ). .. RNA was visualized after ethidium bromide staining under UV light (254 nm) in a gel documentation system (E-Box-3026, peQlab).

    Agarose Gel Electrophoresis:

    Article Title: Light-activated communication in synthetic tissues
    Article Snippet: DNAse I (M0303, NEB) was then added to the tubes, which were held at 37°C for an additional 20 min. EDTA was then added (5 mM final concentration), and the enzymes were denatured at 75°C for 10 min. .. The entire sample was then run on a 2% (w/v) TAE agarose gel with a single-stranded RNA ladder (N0364, NEB). .. PURExpress protein expression in bulk from LA-DNA Protein expression was performed with the PURExpress In Vitro Protein Synthesis kit (E6800, NEB) according to the manufacturer’s protocol with the addition of a murine RNAse inhibitor (MB0314, NEB).

    Molecular Weight:

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs
    Article Snippet: .. Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ). .. RNA was visualized after ethidium bromide staining under UV light (254 nm) in a gel documentation system (E-Box-3026, peQlab).

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    New England Biolabs low range ssrna ladder
    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range <t>ssRNA</t> Ladder (NEB); MF, <t>RiboRuler</t> Low Range RNA Ladder (Thermo Fisher Scientific).
    Low Range Ssrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/low range ssrna ladder/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    low range ssrna ladder - by Bioz Stars, 2021-05
    95/100 stars
      Buy from Supplier

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    Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Journal: Nucleic Acids Research

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs

    doi: 10.1093/nar/gkw786

    Figure Lengend Snippet: Incubation of in vitro transcripts of CRISPR1, 2 and 3 with Cas6-1. ( A ) A total of 2.3 μM of CRISPR1 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( B ) A total of 2 μM of CRISPR2 transcript is cleaved by 6.2 μM Cas6-1 by incubation for 1 h. ( C ) A total of 3.2 μM of CRISPR3 transcript is not cleaved by 6.5 μM Cas6-1 by incubation for 2 h. ( D ) The cleavage of 6.6 μM of the shorter CRISPR1* in vitro transcript by 5.8 μM Cas6-1 monitored over a time of 2 h 20 min. Since no finally mature crRNA ( 17 ) was detected, the in vivo presence of an unknown ribonuclease is suggested. All reactions were performed at 37°C in a reaction volume of 5 μl. RNA was separated by 8 M urea 10% PAGE and bands were visualized by ethidium bromide staining. Diamonds represent repeats and rectangles spacer sequences. Transcripts were not gel purified, explaining the appearance of fragments smaller than the full length transcripts of CRISPR1 and 2 in absence of Cas6-1. Eb1, elution buffer 1 used as negative control. Molecular markers: MN, Low Range ssRNA Ladder (NEB); MF, RiboRuler Low Range RNA Ladder (Thermo Fisher Scientific).

    Article Snippet: Before loading onto denaturing 8 M urea 10% PAA gels, reactions were incubated at 95°C for 5 min. As size markers served the Low Range ssRNA Ladder from NEB (MN ), the RiboRuler Low Range RNA Ladder from Thermo Fisher Scientific (MF ) and the Low Molecular Weight Marker from Affymetrix (MA ).

    Techniques: Incubation, In Vitro, In Vivo, Polyacrylamide Gel Electrophoresis, Staining, Purification, Negative Control

    Ribonuclease activity in the presence of EDTA and divalent cations. A. MBP-RNase I ribonuclease activity assay. The low range ssRNA ladder (50 to 1000 nt long, NEB) was used as the substrate for RNase activity assay in a high sale buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) supplemented with divalent cations (1 mM) or EDTA (10 mM).

    Journal: bioRxiv

    Article Title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

    doi: 10.1101/2021.01.31.429007

    Figure Lengend Snippet: Ribonuclease activity in the presence of EDTA and divalent cations. A. MBP-RNase I ribonuclease activity assay. The low range ssRNA ladder (50 to 1000 nt long, NEB) was used as the substrate for RNase activity assay in a high sale buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5) supplemented with divalent cations (1 mM) or EDTA (10 mM).

    Article Snippet: To demonstrate the ribonuclease activity for the partially purified MBP-RNase I, we incubated a low range ssRNA ladder (N0364S, NEB) with the enzyme (3 enzyme dilutions at 2 μg, 0.2 μg, and 0.02 μg) in a high salt buffer supplemented with EDTA, Ni2+ , Ca2+ , Mn2+ , Co2+ , or Mg2+ .

    Techniques: Activity Assay

    Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Journal: Nucleic Acids Research

    Article Title: In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

    doi: 10.1093/nar/gku120

    Figure Lengend Snippet: Cascade assembly and RNA binding. ( A ) The Cascade subunits (Csa5, Cas7, Cas5a, Cas3′, Cas3′′ and Cas8a2) were assembled via a co-refolding procedure of insoluble recombinant proteins, incubated with synthetic crRNA 5.2 (crRNA) or no RNA (−RNA), and protein interaction was verified via size-exclusion chromatography. In all, 15% SDS–PAGE of individual fractions (fractions 10–18) shows distinct protein bands of assembled Cas protein subunits. The Cascade-containing fractions were additionally analyzed for bound RNA content by 10% Urea-PAGE. A protein-free sample served as a running control (−Cascade). ( B ) A comparison of the EMSAs for crRNA binding by the individual Cas7 subunit (lanes 1–7: 0, 0.2, 0.5, 1, 2, 4, 5 µM) or Cas7 assembled into Cascade (lanes 8–10: 0.5, 2, 5 µM) on 1% agarose gels demonstrates the formation of high shifts for Cas7 alone and lower, more diffuse shifts for Cascade. ( C ) EMSAs verified the binding of the 5′-[γ- 32 P]-ATP labeled crRNA 5.13 by Cascade (with Cas7 purified via a SUMO-fused construct) in increasing concentrations (lanes 1–6: 0, 0.125, 0.25, 0.5, 1, 2 µM) and in the presence of yeast RNA via 6% native PAGE.

    Article Snippet: The cleaved crRNA was purified by phenol/chloroform extraction (pH 5.2), EtOH precipitated with the addition of glycogen (1:100, v/v), mixed with 2× formamide loading buffer (95% formamide, 5 mM EDTA, pH 8.0, 2.5 mg bromophenol blue, 2.5 mg xylene cyanol), heated for 5 min at 95°C, separated by a denaturing-PAGE (8 M urea, 1× TBE, 10% polyacrylamide) next to an RNA marker (low range ssRNA ladder, NEB) and visualized by toluidine blue staining.

    Techniques: RNA Binding Assay, Recombinant, Incubation, Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Binding Assay, Labeling, Purification, Construct, Clear Native PAGE