murine rnase inhibitor  (New England Biolabs)


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    Structured Review

    New England Biolabs murine rnase inhibitor
    Murine Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/murine rnase inhibitor/product/New England Biolabs
    Average 93 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    murine rnase inhibitor - by Bioz Stars, 2020-05
    93/100 stars

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    Centrifugation:

    Article Title: Combination of Four Serum Exosomal MiRNAs as Novel Diagnostic Biomarkers for Early-Stage Gastric Cancer
    Article Snippet: .. Afterward, murine RNase inhibitor (NEB, Ipswich, MA, United States) was added to the mixture (150 units/ml) before centrifugation at 1500 g for 30 min. .. Finally, the supernatants were aspirated and the pelleted exosomes were re-suspended in 50 μl 1 × PBS, and immediately used for RNA extraction.

    Ligation:

    Article Title: Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.
    Article Snippet: .. For linker ligation, a maximum of 5 pmol RNA in 5 ml were denatured for 2 min at 80 C before 8 ml 50% sterile filtered PEG MW 8000, 2 ml DMSO, 2 ml 10x T4 RNA Ligase 2 buffer (NEB), 1 ml murine RNase inhibitor, 1 ml 1 mg/ml linker L1 and 1 ml truncated T4 RNALigase 2 (NEB) were added and incubated for 2.5 hr at 37 C or 23 C. Nucleic acids were precipitated as described before and resuspended in 6 ml 10mMTris-HCl pH 7.0. .. Samples were run on a 10%TBE-Urea polyacrylamide gel (Invitrogen) in 1x TBE (Ambion) for 50min at 200 V. Gels were stained for 20min with SYBR gold and desired gel pieces were excised and RNA was extracted as described before.

    Purification:

    Article Title: RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2
    Article Snippet: .. After purification, RNAs were labelled with NHS-Cyanin-5 (Kerafast) in 70 mM NaHCO3 pH 8.8 with murine RNase inhibitor (NEB) for 2 h at RT. ..

    Article Title: A direct role for SNX9 in the biogenesis of filopodia
    Article Snippet: .. 1 µg purified linear DNA was added to a standard mMESSAGE mMACHINE transcription reaction with the addition of 2 U/µl of Murine RNase inhibitor (New England Biolabs) and incubated for 2 h at 37°C. .. Unincorporated nucleotides were removed using the RNeasy mini kit (Qiagen).

    Concentration Assay:

    Article Title: Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro
    Article Snippet: .. 80 µL of DMS reaction buffer (100 mM KCl, 40 mM HEPES pH 7.5, 0.5 mM MgCl2 ) and 100 U of Murine Rnase Inhibitor (NEB) were added, followed by incubation with mixing at 25 °C for 5 min. Next, DMS was added to the final concentration of 5%, and samples were incubated for 6 min at 25 °C with gentle mixing. ..

    Incubation:

    Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.
    Article Snippet: .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin.

    Article Title: Profiling Ssb-Nascent Chain Interactions Reveals Principles of Hsp70-Assisted Folding.
    Article Snippet: .. For linker ligation, a maximum of 5 pmol RNA in 5 ml were denatured for 2 min at 80 C before 8 ml 50% sterile filtered PEG MW 8000, 2 ml DMSO, 2 ml 10x T4 RNA Ligase 2 buffer (NEB), 1 ml murine RNase inhibitor, 1 ml 1 mg/ml linker L1 and 1 ml truncated T4 RNALigase 2 (NEB) were added and incubated for 2.5 hr at 37 C or 23 C. Nucleic acids were precipitated as described before and resuspended in 6 ml 10mMTris-HCl pH 7.0. .. Samples were run on a 10%TBE-Urea polyacrylamide gel (Invitrogen) in 1x TBE (Ambion) for 50min at 200 V. Gels were stained for 20min with SYBR gold and desired gel pieces were excised and RNA was extracted as described before.

    Article Title: A direct role for SNX9 in the biogenesis of filopodia
    Article Snippet: .. 1 µg purified linear DNA was added to a standard mMESSAGE mMACHINE transcription reaction with the addition of 2 U/µl of Murine RNase inhibitor (New England Biolabs) and incubated for 2 h at 37°C. .. Unincorporated nucleotides were removed using the RNeasy mini kit (Qiagen).

    Article Title: Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro
    Article Snippet: .. 80 µL of DMS reaction buffer (100 mM KCl, 40 mM HEPES pH 7.5, 0.5 mM MgCl2 ) and 100 U of Murine Rnase Inhibitor (NEB) were added, followed by incubation with mixing at 25 °C for 5 min. Next, DMS was added to the final concentration of 5%, and samples were incubated for 6 min at 25 °C with gentle mixing. ..

    Fractionation:

    Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.
    Article Snippet: .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin.

    Cross-linking Immunoprecipitation:

    Article Title: Distinct Classes of Chromatin Loops Revealed by Deletion of an RNA-Binding Region in CTCF.
    Article Snippet: .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin. .. Mammalian genomes are folded into topologically associating domains (TADs), consisting of chromatin loops anchored by CTCF and cohesin.

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    New England Biolabs murine rnase inhibitor
    Biochemical characterization of LwaCas13a <t>RNA</t> cleavage activity a, LwaCas13a has more active <t>RNAse</t> activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.
    Murine Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/murine rnase inhibitor/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    murine rnase inhibitor - by Bioz Stars, 2020-05
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      Buy from Supplier

    99
    New England Biolabs rnase a inhibitor
    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with <t>RNAse</t> A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.
    Rnase A Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase a inhibitor/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

    Figure Lengend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Article Snippet: Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR

    eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF3d cap-binding activity is required for efficient 48S initiation complex formation on specific mRNAs a , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to eIF3 in the presence of competitor ligands. b , Electrostatic surface view of the eIF3d cap-binding domain colored by charge, with a zoomed view of single stranded RNA (ssRNA) and cap analog modeled according to their positions bound to DXO 15 . Positive charge is colored blue and negative charge is in red, and the RNA gate is removed for clarity. c , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled c-Jun 5′ UTR RNA crosslinked to wild type or helix α5 or helix α11-mutant eIF3. eIF3d-helix α5 mutant (D249Q/V262I/Y263A), helix α11 mutant (T317E/N320E/H321A). WT, wild type. d , Incorporation of c-Jun and ACTB mRNA into initiation complexes by wild type, helix α5, or helix α11-mutant eIF3d as measured by quantitative RT-PCR. mRNA-ribosome association is expressed as the ratio between the quantity of mRNA transcripts to 18S rRNA and normalized to the wild type sample. The results are representative of three independent experiments and given as the mean ± s.d. from a representative quantitative RT-PCR experiment performed in duplicate.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Binding Assay, Activity Assay, SDS-Gel, Labeling, Mutagenesis, Quantitative RT-PCR

    eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: eIF4E recognizes the 5′ end of the c-Jun mRNA less efficiently than ACTB mRNA a , Coomassie blue stained SDS gel of recombinant human eIF4E expressed in E. coli. b , Phosphorimage of SDS gel resolving RNase-protected 32 P-cap-labeled ACTB or c-Jun 5′ UTR RNA crosslinked to eIF4E. The result is representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: Staining, SDS-Gel, Recombinant, Labeling

    5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Journal: Nature

    Article Title: eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    doi: 10.1038/nature18954

    Figure Lengend Snippet: 5' end recognition of c-Jun mRNA is eIF4F-independent a , Distribution of c-Jun or ACTB mRNA-containing initiation complexes in programmed 293T cell in vitro translation extracts. The mRNA abundance (black line) is expressed as the fraction of total recovered transcripts. The results are given as the mean ± standard deviation (s.d.) of a representative quantitative RT-PCR experiment performed in duplicate. The polysome profile (gray line) is plotted as relative absorbance at 254 nm versus elution fractions. b , Western blot analysis of initiation factors in 48S translation complexes formed on c-Jun and ACTB mRNAs. 293T, total protein from 293T in vitro translation extracts. For gel source data, see Supplementary Figure 1 . c , Phosphorimage of SDS gel resolving RNase-protected 32 P-internal or 32 P-cap-labeled c-Jun 5' UTR RNA crosslinked to eIF3 subunits. Recombinant eIF3a migrates at ~100 kDa due to a C-terminal truncation 26 . The results of a - c are representative of three independent experiments.

    Article Snippet: Each translation reaction contained 50% in vitro translation lysate and buffer to make the final reaction with 0.84 mM ATP, 0.21 mM GTP, 21 mM creatine phosphate (Roche), 45 U ml-1 creatine phosphokinase (Roche), 10 mM HEPES-KOH pH 7.6, 2 mM DTT, 8 mM amino acids (Promega), 255 mM spermidine, 1 U ml-1 murine RNase inhibitor (NEB), and mRNA-specific concentrations of Mg(OAc)2 and KOAc.

    Techniques: In Vitro, Standard Deviation, Quantitative RT-PCR, Western Blot, SDS-Gel, Labeling, Recombinant

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Journal: Cell

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    doi: 10.1016/j.cell.2016.12.020

    Figure Lengend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Article Snippet: Reactions contained 1× HAT assay buffer (50mM Tris-HCl pH 7.5 (RT), 5% glycerol, 0.1mM EDTA, 50mM KCl), 1mM DTT, 10mM Na-Butyrate, 1× Complete EDTA protease inhibitor cocktail (Roche), 0.4U/ul murine RNAse inhibitor (NEB), 0.1mg/ml BSA (NEB), 80nM H31–21 peptide (Anaspec) and the required dilution of RNA probe.

    Techniques: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test

    Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: Effect of the growth parameters on RiCF. (A) Schematics of a hypothetical scenario when RNA inhibits NAPs that could potentially cleave DNA. During lysis, quick RNA degradation removes the inhibition resulting in breakage of chromosomes. (B) Growth phase dependence of RiCF. AB1157 was grown at 37°C with periodic OD measurements, and samples for plugs were withdrawn at various times. The cells were made into plugs using lysis agarose and RNase (50 μg/plug) and the plugs were lysed and electrophoresed under standard conditions. Data points are means of at least three independent assays ± SEM. (C) Effect of translation and transcription inhibition on RiCF. Cells were grown till OD 0.5–0.6, split into three parts and chloramphenicol (40 μg/ml) or rifampicin (150 μg/ml) were added to two samples. All sample were shaken for another 2–3 hours at 37°C before making plugs as described in (B). Data points are means of four independent assays ± SEM. (D) Growth in minimal medium reduces RiCF. Cells were grown in LB or MOPS till the OD reached 0.6 and made into plugs using standard conditions. The values presented are means of six independent assays ± SEM. (E) Effect of growth temperature on RNase-induced chromosomal fragmentation. Cultures of AB1157 were grown at 20°C, 30°C, 37°C, 42°C or 45°C to same cell densities (A 600 = 0.6), and plugs were made in lysis agarose with RNAse A (50 μg/plug), as described in (A). Data are means of three to six independent measurements ± SEM.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Lysis, Inhibition

    RNA degradation causes chromosomal fragmentation. (A)  Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation.  (B)  Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C.  (C)  Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE.  (D)  A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f  and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug.  (E)  Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Journal: PLoS ONE

    Article Title: Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome

    doi: 10.1371/journal.pone.0190177

    Figure Lengend Snippet: RNA degradation causes chromosomal fragmentation. (A) Schematics of a hypothetical scenario when RNA makes the central core of nucleoids, and its degradation results in collapse of the nucleoid structure, causing chromosomal fragmentation. (B) Radiogram of a pulsed field gel showing chromosomal fragmentation in AB1157 when cells were embedded in agarose plugs in the presence and absence of proteinase K (25 μg/plug) and/or RNase (50 μg/plug) and lysed overnight at 62°C. (C) Radiogram showing DNase I sensitivity of the signal entering the gel. Plugs were lysed at 62°C, washed extensively to remove traces of lysis buffer and then treated with DNase I at 37°C before PFGE. (D) A representative gel showing that RNA degradation by different enzymes causes chromosomal fragmentation. Plugs were made in the absence of proteinase K in 1x restriction enzyme buffer (NEBuffer 3 for RNase A, XRN-1 and RNAse I f and NEBuffer 4 for Exo T). The concentrations of the enzymes used were, RNase, 50 μg/plug; XRN-1, 5 U/plug; RNAse I f , 100 U/plug and Exo T, 20 U/plug. (E) Quantification of the chromosomal fragmentation when plugs were made in the presence of various RNA degrading enzymes. The values presented are means of four independent assays ± SEM. CZ, compression zone.

    Article Snippet: XRN-I, RNase If , Exonuclease T (Exo T), EcoRI and RNase A inhibitor were all from New England Biolabs.

    Techniques: Pulsed-Field Gel, Lysis