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
    Size:
    100 gel lanes
    Category:
    RNA Ladders
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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 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    low range ssrna ladder - by Bioz Stars, 2020-04
    99/100 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

    Related Articles

    Synthesized:

    Article Title: Multiplexed miRNA northern blots via hybridization chain reaction
    Article Snippet: LNA probes used for the sensitivity/selectivity comparisons of Supplementary Sections S5 and 6 were synthesized by Exiqon using catalog detection sequences with a DNA HCR initiator at each end. .. The low range ssRNA ladder and microRNA marker were purchased from New England Biolabs (catalog #N0364 and #N2102).

    Article Title: Detection of Endogenous MazF Enzymatic Activity in Staphylococcus aureus
    Article Snippet: Primers and oligonucleotides were synthesized by IDT. .. Restriction enzymes, BSA, Low-Range ssRNA Ladder, RNase Inhibitor—Human Placenta, and E. coli strains DH5α and NiCo21(DE3) [ ] were purchased from New England Biolabs (NEB).

    Electrophoresis:

    Article Title: The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei
    Article Snippet: The reaction products were separated by electrophoresis on 10% polyacrylamide gel containing 7M urea and 1X TBE, transferred to Whatman paper, dried, and then visualized after exposure to PhosphorImager screen (GE Healthcare). .. Labeled gRNAs were identified by size in comparison to the labeled low range ssRNA ladder (NEB).

    Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
    Article Snippet: Northern blot analysis For electrophoresis 15% polyacrylamide gels containing 8 M urea and 0.5x MOPS running buffer (10x MOPS is 200 mM MOPS, 50 mM sodium acetate and 10 mM EDTA, pH 7.0) were used. .. In addition 1 μl of microRNA ladder (NEB) and 5 μl of low range ssRNA ladder (NEB) were loaded onto the gel.

    Incubation:

    Article Title: The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei
    Article Snippet: 1 µg of treated RNA was incubated at 37°C for 1 h in a 15 µl reaction containing 40 µCi [α-32 P]GTP (3000 Ci/mmol) and 10U of guanylyltransferase (Epicentre Biotechnologies) according to the manufacturer's instructions. .. Labeled gRNAs were identified by size in comparison to the labeled low range ssRNA ladder (NEB).

    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).

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: To determine the cleavage rate constant kcl of a first order reaction 500 nM CRISPR1 repeat were incubated in different ratios with Cas6-1 (2:1, 4:1, 8:1 with 1 µM, 2 µM and 4 µM enzyme) for indicated time points up to 15 min. Fractions of substrate and product were determined from the gel images using Quantity One software (BIO-RAD). .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Article Title: Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes
    Article Snippet: U1/5.8s-rRNA and U4 snRNAs were purified by cutting out the 164nt and 141nt bands respectively, using the low-range ssRNA ladder as a size marker (NEB N0364). snRNAs were gel eluted in elution buffer [0.4 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA pH 8] at 16 °C overnight with shaking at 2000 rpm. .. The reaction was supplemented with 2 µl 1U µl−1 Nuclease P1 (Sigma N8630) in a 30-µl reaction with 0.2 mM ZnCl2 and 20 mM NH4 OAc pH 5.3, incubated for 2 hr at 42 °C.

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: Split 1 RNA, Split 2 RNA and complementary Split 1/Split 2 DBS were mixed in a 1:1:1 ratio and incubated together at 37 °C for 25 min. All the samples including negative controls (Split sequences without complementary DBS) were run on 6% NovexTM TBE gel in 1x TBE buffer at 200 V for 45 min. In-gel imaging was performed as previously described . .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
    Article Snippet: In short, equal amounts of total RNA (5 μg to 25 μg per lane) in a volume of 4 μl were mixed with 11 μl FDF buffer (2.75 μl formaldehyde (37%), 7.5 μl formamide ( > 99.5%) and 0.75 μl 10x MOPS buffer), incubated at 55°C for 15 min and subsequently mixed with 2 μl of 10x dyes (0.05% [w/v] xylene cyanol and 0.05% [w/v] bromphenol blue in nuclease-free water) before loading. .. In addition 1 μl of microRNA ladder (NEB) and 5 μl of low range ssRNA ladder (NEB) were loaded onto the gel.

    Activity Assay:

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: To test whether the catalytic activity of the Cas6-1 H51A mutant can be restored, the cleavage buffer was supplemented with 500 mM imidazole, pH 8.0 and either pre-incubated at 4°C overnight or directly transferred to 30°C. .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Hybridization:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well. .. The RNA was crosslinked to the membrane by exposure to UV light, washed in 0.1X SSC and 0.1% sodium dodecyl sulfate (SDS) at 65°C for 1 h and prehybridized for 1 h in hybridization solution (6X SSC, 10X Denhardts solution (Life Technologies), 0.1% SDS) at 42°C.

    Article Title: Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus
    Article Snippet: Northern blot analysis 2–5 μg total RNA per lane was denatured for 10 min at 65°C in the loading buffer containing 95% (v/v) formamide and separated on 8% polyacrylamide gels containing 7.6 M urea with a low range ssRNA ladder (New England Biolabs). .. Membranes were prehybridized at 42°C, followed by hybridization with 10 pmol 5′-biotin-labeled DNA probes ( ) for 12 h. After three rounds of washing for 10 min each in 1×, 0.2×, and 0.1× SSC–0.1% SDS solutions, signals were visualized using chemiluminescent nucleic acid detection module (Thermo Scientific) according to the manufacturer's protocol.

    High Performance Liquid Chromatography:

    Article Title: Multiplexed miRNA northern blots via hybridization chain reaction
    Article Snippet: The low range ssRNA ladder and microRNA marker were purchased from New England Biolabs (catalog #N0364 and #N2102). .. Synthetic miRNA targets used for sensitivity and selectivity studies were purchased 5′-phosphorylated and HPLC-purified from Integrated DNA Technologies (IDT).

    Transfection:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: Cells were harvested 48 h after transfection and total RNA was extracted using the RNeasy (Qiagen) kit according to manufacturer recommendations. .. 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well.

    Northern Blot:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: Paragraph title: Northern blotting ... 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well.

    Article Title: Global mapping transcriptional start sites revealed both transcriptional and post-transcriptional regulation of cold adaptation in the methanogenic archaeon Methanolobus psychrophilus
    Article Snippet: .. Northern blot analysis 2–5 μg total RNA per lane was denatured for 10 min at 65°C in the loading buffer containing 95% (v/v) formamide and separated on 8% polyacrylamide gels containing 7.6 M urea with a low range ssRNA ladder (New England Biolabs). .. After separation, RNAs were transferred onto Hybond-N+ membranes (GE Healthcare) by electroblotting and cross-linked to the membrane using UV.

    Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
    Article Snippet: Paragraph title: Northern blot analysis ... In addition 1 μl of microRNA ladder (NEB) and 5 μl of low range ssRNA ladder (NEB) were loaded onto the gel.

    Generated:

    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 ). .. For size determination of RNA fragments generated in cleavage assays with Cas6-1 or Cas6-2a separation of fragments was performed with a sequencing gel electrophoresis apparatus (Model S2, Biometra).

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation. .. The CRISPR1 repeat oligo ladder (C1L) was generated by alkaline hydrolysis (50 mM Tris-HCl pH 8.5, 20 mM MgCl2 ) with 25 pmol substrate and was incubated for 72 h to 96 h at 30°C.

    Imaging:

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: Paragraph title: Assembly of Split sequences with co mplementary DBS and in-gel imaging ... The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Polymerase Chain Reaction:

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: The PCR product was purified using Monarch® PCR & DNA Cleanup kit (NEB) and the DNA concentration was measured on a NanoDrop spectrophotometer. .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    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).

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: .. The low range ssRNA ladder (NEB) was used as molecular weight marker. .. The set of staple strands (see Supplementary Table ) were mixed in 10-fold excess with RNA scaffold in 50 μL of folding buffer (10 mM MgCl2 , 20 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0) .

    Nucleic Acid Electrophoresis:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: All solutions for gel electrophoresis and Northern blotting were made in diethylpyrocarbonate (DEPC)-treated and autoclaved water. .. 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well.

    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 ). .. For size determination of RNA fragments generated in cleavage assays with Cas6-1 or Cas6-2a separation of fragments was performed with a sequencing gel electrophoresis apparatus (Model S2, Biometra).

    Mutagenesis:

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: To test whether the catalytic activity of the Cas6-1 H51A mutant can be restored, the cleavage buffer was supplemented with 500 mM imidazole, pH 8.0 and either pre-incubated at 4°C overnight or directly transferred to 30°C. .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Isolation:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well. .. The gel was run in running buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA pH 8.0, 6.3% formaldehyde) at 60 V for 2.5 h. To assess the quality of the isolated RNA, the gel was then stained with ethidium bromide (10 μL of 10 mg/mL ethidium bromide in 400 mL running buffer).

    Article Title: Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes
    Article Snippet: Paragraph title: snRNA isolation and nucleoside UHPLC-MS/MS ... U1/5.8s-rRNA and U4 snRNAs were purified by cutting out the 164nt and 141nt bands respectively, using the low-range ssRNA ladder as a size marker (NEB N0364). snRNAs were gel eluted in elution buffer [0.4 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA pH 8] at 16 °C overnight with shaking at 2000 rpm.

    Subcloning:

    Article Title: Detection of Endogenous MazF Enzymatic Activity in Staphylococcus aureus
    Article Snippet: Restriction enzymes, BSA, Low-Range ssRNA Ladder, RNase Inhibitor—Human Placenta, and E. coli strains DH5α and NiCo21(DE3) [ ] were purchased from New England Biolabs (NEB). .. Subcloning Efficiency DH5α Chemically Competent E. coli was purchased from Invitrogen.

    Labeling:

    Article Title: The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei
    Article Snippet: .. Labeled gRNAs were identified by size in comparison to the labeled low range ssRNA ladder (NEB). ..

    Purification:

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: Paragraph title: RNA scaffold synthesis and purification ... The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Article Title: Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes
    Article Snippet: .. U1/5.8s-rRNA and U4 snRNAs were purified by cutting out the 164nt and 141nt bands respectively, using the low-range ssRNA ladder as a size marker (NEB N0364). snRNAs were gel eluted in elution buffer [0.4 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA pH 8] at 16 °C overnight with shaking at 2000 rpm. ..

    Sequencing:

    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 ). .. For size determination of RNA fragments generated in cleavage assays with Cas6-1 or Cas6-2a separation of fragments was performed with a sequencing gel electrophoresis apparatus (Model S2, Biometra).

    Software:

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: To determine the cleavage rate constant kcl of a first order reaction 500 nM CRISPR1 repeat were incubated in different ratios with Cas6-1 (2:1, 4:1, 8:1 with 1 µM, 2 µM and 4 µM enzyme) for indicated time points up to 15 min. Fractions of substrate and product were determined from the gel images using Quantity One software (BIO-RAD). .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    In Vitro:

    Article Title: Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs
    Article Snippet: Paragraph title: RNase cleavage assays with in vitro transcripts ... 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 ).

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: Paragraph title: RNase cleavage assays with synthetic oligonucleotides or in vitro transcripts ... The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: After the addition of the RNA loading dye 2x (NEB) and the heat denaturation at 65 °C for 5 min, in vitro -transcribed RNA was loaded and run on 10% TBE-Urea gel in 1x TBE buffer at 200 V for 40 min. After staining with SYBR® Gold in 1x TBE for 8 min, the gels were visualized using Typhoon laser scanner. .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Spectrophotometry:

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: The noDBS1 and noDBS2 scaffold sequences were transcribed in vitro at 37 °C for 2 h. After DNase treatment at 37 °C for 15 min, the RNA transcript was purifed using RNA Clean & ConcentratorTM (Zymo Research) and quantified using a NanoDrop spectrophotometer. .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Concentration Assay:

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: The PCR product was purified using Monarch® PCR & DNA Cleanup kit (NEB) and the DNA concentration was measured on a NanoDrop spectrophotometer. .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Marker:

    Article Title: Multiplexed miRNA northern blots via hybridization chain reaction
    Article Snippet: .. The low range ssRNA ladder and microRNA marker were purchased from New England Biolabs (catalog #N0364 and #N2102). .. Synthetic miRNA targets used for sensitivity and selectivity studies were purchased 5′-phosphorylated and HPLC-purified from Integrated DNA Technologies (IDT).

    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).

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803
    Article Snippet: .. The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation. .. The CRISPR1 repeat oligo ladder (C1L) was generated by alkaline hydrolysis (50 mM Tris-HCl pH 8.5, 20 mM MgCl2 ) with 25 pmol substrate and was incubated for 72 h to 96 h at 30°C.

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: .. The low range ssRNA ladder (NEB) was used as molecular weight marker. .. The set of staple strands (see Supplementary Table ) were mixed in 10-fold excess with RNA scaffold in 50 μL of folding buffer (10 mM MgCl2 , 20 mM Tris-HCl pH 7.6, 1 mM EDTA pH 8.0) .

    Article Title: Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes
    Article Snippet: .. U1/5.8s-rRNA and U4 snRNAs were purified by cutting out the 164nt and 141nt bands respectively, using the low-range ssRNA ladder as a size marker (NEB N0364). snRNAs were gel eluted in elution buffer [0.4 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA pH 8] at 16 °C overnight with shaking at 2000 rpm. ..

    Staining:

    Article Title: A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells
    Article Snippet: 10 μL of a Low Range ssRNA Ladder (New England Biolabs) was loaded as well. .. The gel was run in running buffer (50 mM sodium acetate pH 5.2, 10 mM EDTA pH 8.0, 6.3% formaldehyde) at 60 V for 2.5 h. To assess the quality of the isolated RNA, the gel was then stained with ethidium bromide (10 μL of 10 mg/mL ethidium bromide in 400 mL running buffer).

    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).

    Article Title: Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon
    Article Snippet: After the addition of the RNA loading dye 2x (NEB) and the heat denaturation at 65 °C for 5 min, in vitro -transcribed RNA was loaded and run on 10% TBE-Urea gel in 1x TBE buffer at 200 V for 40 min. After staining with SYBR® Gold in 1x TBE for 8 min, the gels were visualized using Typhoon laser scanner. .. The low range ssRNA ladder (NEB) was used as molecular weight marker.

    Article Title: Atlas of quantitative single-base-resolution N6-methyl-adenine methylomes
    Article Snippet: snRNA isolation and nucleoside UHPLC-MS/MS Nuclear-enriched RNA was resolved on a 6% TBE-urea gel then stained with SYBR-gold (Invitrogen S11494). .. U1/5.8s-rRNA and U4 snRNAs were purified by cutting out the 164nt and 141nt bands respectively, using the low-range ssRNA ladder as a size marker (NEB N0364). snRNAs were gel eluted in elution buffer [0.4 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA pH 8] at 16 °C overnight with shaking at 2000 rpm.

    Article Title: Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation
    Article Snippet: In addition 1 μl of microRNA ladder (NEB) and 5 μl of low range ssRNA ladder (NEB) were loaded onto the gel. .. After electrophoresis, gels were stained in ethidium bromide (10 μg/ml) to visualize the RNA size markers.

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    New England Biolabs rna marker
    Cascade assembly and <t>RNA</t> 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% <t>SDS–PAGE</t> 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.
    Rna Marker, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna marker/product/New England Biolabs
    Average 90 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    rna marker - by Bioz Stars, 2020-04
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    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

    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

    Loss of KREPA3 or mutation of KREPA3 ZF2 has no effect on gRNA levels. Total cellular RNA from KREPA3-RKO, RKO-KREPA3 WT-myc and ZFm2-myc cells with KREP3 Reg expressed (E) or repressed (R) were examined by a capping assay using guanylyltransferse and [α- 32 P]GTP. The similarly labeled ssRNA ladder was used to size the gRNAs which show their characteristics size heterogeneity due to their variable oligo-U tails.

    Journal: PLoS ONE

    Article Title: The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei

    doi: 10.1371/journal.pone.0008913

    Figure Lengend Snippet: Loss of KREPA3 or mutation of KREPA3 ZF2 has no effect on gRNA levels. Total cellular RNA from KREPA3-RKO, RKO-KREPA3 WT-myc and ZFm2-myc cells with KREP3 Reg expressed (E) or repressed (R) were examined by a capping assay using guanylyltransferse and [α- 32 P]GTP. The similarly labeled ssRNA ladder was used to size the gRNAs which show their characteristics size heterogeneity due to their variable oligo-U tails.

    Article Snippet: Labeled gRNAs were identified by size in comparison to the labeled low range ssRNA ladder (NEB).

    Techniques: Mutagenesis, Labeling

    Purification and in vitro cleavage assay of Synechocystis WT Cas6-1 and mutated Cas6-1 proteins. (A) Coomassie-stained 15% SDS-PAGE of heterologously overexpressed and purified Cas6-1 protein variants. The expected molecular weight of Cas6-1_6xHis is ∼ 30 kDa. 66.67 pmol recombinant enzyme was loaded on each lane and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) served for protein size estimation. (B, C) In in vitro cleavage assays both the (B) CRISPR1 RNA oligo repeat sequence and the (C) pre-acrRNA cassette is processed with variable efficiency by the purified Cas6-1 protein variants shown in (A). After incubation at 30°C the RNA fragments were electrophoretically size separated on a 15% PAA sequencing gel and visualized with SYBR® Gold Nucleic Acid Gel Stain. The processing results in a pattern of RNA fragments of distinct lengths. Cleavage assays were conducted with a three-fold excess (B) or a 1:1 ratio (C) of protein to RNA-substrate in cleavage buffer and incubated at 30°C for the indicated time. Expected RNA fragments and their corresponding sizes are illustrated by cartoons. Another Cas6-1 WT protein sample, which was overexpressed and purified independently, is indicated by an asterisk. Negative controls were complemented with 1 μL of H 2 O or 1 μL eluate of a mock control. Two additional nucleotides resulting from in vitro transcription were added to the 215 nt long acrRNA yielding a 217 nt long pre-acrRNA. For RNA size estimation a CRISPR1 repeat oligo ladder (C1L), RiboRuler low range RNA ladder (Thermo Fisher Scientific), low range ssRNA ladder or microRNA marker (both from New England Biolabs) were used.

    Journal: RNA Biology

    Article Title: Biochemical analysis of the Cas6-1 RNA endonuclease associated with the subtype I-D CRISPR-Cas system in Synechocystis sp. PCC 6803

    doi: 10.1080/15476286.2018.1447742

    Figure Lengend Snippet: Purification and in vitro cleavage assay of Synechocystis WT Cas6-1 and mutated Cas6-1 proteins. (A) Coomassie-stained 15% SDS-PAGE of heterologously overexpressed and purified Cas6-1 protein variants. The expected molecular weight of Cas6-1_6xHis is ∼ 30 kDa. 66.67 pmol recombinant enzyme was loaded on each lane and PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific) served for protein size estimation. (B, C) In in vitro cleavage assays both the (B) CRISPR1 RNA oligo repeat sequence and the (C) pre-acrRNA cassette is processed with variable efficiency by the purified Cas6-1 protein variants shown in (A). After incubation at 30°C the RNA fragments were electrophoretically size separated on a 15% PAA sequencing gel and visualized with SYBR® Gold Nucleic Acid Gel Stain. The processing results in a pattern of RNA fragments of distinct lengths. Cleavage assays were conducted with a three-fold excess (B) or a 1:1 ratio (C) of protein to RNA-substrate in cleavage buffer and incubated at 30°C for the indicated time. Expected RNA fragments and their corresponding sizes are illustrated by cartoons. Another Cas6-1 WT protein sample, which was overexpressed and purified independently, is indicated by an asterisk. Negative controls were complemented with 1 μL of H 2 O or 1 μL eluate of a mock control. Two additional nucleotides resulting from in vitro transcription were added to the 215 nt long acrRNA yielding a 217 nt long pre-acrRNA. For RNA size estimation a CRISPR1 repeat oligo ladder (C1L), RiboRuler low range RNA ladder (Thermo Fisher Scientific), low range ssRNA ladder or microRNA marker (both from New England Biolabs) were used.

    Article Snippet: The low range ssRNA ladder (ssRNA marker) (New England Biolabs), the RiboRuler low range RNA ladder (Thermo Fisher Scientific) and the microRNA marker (New England Biolabs) served as RNA ladders for size estimation.

    Techniques: Purification, In Vitro, Cleavage Assay, Staining, SDS Page, Molecular Weight, Recombinant, Sequencing, Incubation, Marker