s1 nuclease  (Thermo Fisher)


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    Name:
    S1 Nuclease
    Description:
    S1 Nuclease is a single-strand-specific endonuclease that hydrolyzes single-stranded RNA or DNA into 5´ mononucleotides. The enzyme will hydrolyze single-stranded regions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65°C.Applications: Nuclease mapping techniques (1,2). Removal of single-stranded regions from double-stranded DNA (3). Exo III-ordered sequencing (4).Source: Isolated from Aspergillus oryzae.Performance and Quality Testing: Double-strand-specific deoxyribonuclease and phosphatase assays.Unit Definition: One unit hydrolyzes 1 µg of denatured DNA to acid-soluble material in 1 min. at 37°C.Unit Reaction Conditions: 30 mM sodium acetate (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 0.5 mg/ml heat-denatured DNA, 5% (v/v) glycerol, and enzyme in 0.5 ml for 10 min. at 37°C.
    Catalog Number:
    18001016
    Price:
    None
    Applications:
    Cloning|DNA & RNA Purification & Analysis|Nuclease Protection Assays|Restriction Enzyme Cloning|Nucleic Acid Gel Electrophoresis & Blotting
    Size:
    20 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher s1 nuclease
    Structural characterization of the DncV reaction product demonstrated a 3′ 3′-linked cGAMP. Digestion of DncV-produced cGAMP with S1 nuclease, RNase T1, and RNase T2 was performed. DncV (5 µM) was incubated with 5 mM ATP + GTP, ATP, or GTP for 1.5 h at 37°C as indicated under A–C. Subsequently, 1 µl of RNase T1, S1 nuclease, or RNase T2 was added for 1 h at 37°C. Products were run on a TLC plate for 30 min at room temperature. (A) 1, ATP + GTP nucleoside triphosphate substrate control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV at 37°C for 1.5 h; 4, ATP + GTP + DncV/S1 nuclease at 37°C for 1.5 h; 5, ATP + GTP + DncV/RNase T1 at 37°C for 1.5 h; 6, ATP + GTP + DncV/RNase T2 at 37°C for 1.5 h. (B) 1, ATP + GTP trinucleotide control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/S1 nuclease; 4, c-di-GMP + S1 nuclease; 5, c-di-AMP + S1 nuclease. (C) 1, ATP + GTP; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/RNase T2; 4, c-di-GMP + RNase T2; 5, c-di-AMP + RNase T2. (D) Expected enzymatic products of cGAMP isomers upon digestion with the RNase enzymes RNase T1 (T1), <t>S1</t> nuclease (S1), and RNase T2 (T2). pG = 5′-GMP, Gp = 3′-GMP, pA = 5′-AMP, Ap = 3′-AMP.
    S1 Nuclease is a single-strand-specific endonuclease that hydrolyzes single-stranded RNA or DNA into 5´ mononucleotides. The enzyme will hydrolyze single-stranded regions in duplex DNA such as loops and gaps. S1 Nuclease is stable at 65°C.Applications: Nuclease mapping techniques (1,2). Removal of single-stranded regions from double-stranded DNA (3). Exo III-ordered sequencing (4).Source: Isolated from Aspergillus oryzae.Performance and Quality Testing: Double-strand-specific deoxyribonuclease and phosphatase assays.Unit Definition: One unit hydrolyzes 1 µg of denatured DNA to acid-soluble material in 1 min. at 37°C.Unit Reaction Conditions: 30 mM sodium acetate (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 0.5 mg/ml heat-denatured DNA, 5% (v/v) glycerol, and enzyme in 0.5 ml for 10 min. at 37°C.
    https://www.bioz.com/result/s1 nuclease/product/Thermo Fisher
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    s1 nuclease - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "DncV Synthesizes Cyclic GMP-AMP and Regulates Biofilm Formation and Motility in Escherichia coli ECOR31"

    Article Title: DncV Synthesizes Cyclic GMP-AMP and Regulates Biofilm Formation and Motility in Escherichia coli ECOR31

    Journal: mBio

    doi: 10.1128/mBio.02492-18

    Structural characterization of the DncV reaction product demonstrated a 3′ 3′-linked cGAMP. Digestion of DncV-produced cGAMP with S1 nuclease, RNase T1, and RNase T2 was performed. DncV (5 µM) was incubated with 5 mM ATP + GTP, ATP, or GTP for 1.5 h at 37°C as indicated under A–C. Subsequently, 1 µl of RNase T1, S1 nuclease, or RNase T2 was added for 1 h at 37°C. Products were run on a TLC plate for 30 min at room temperature. (A) 1, ATP + GTP nucleoside triphosphate substrate control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV at 37°C for 1.5 h; 4, ATP + GTP + DncV/S1 nuclease at 37°C for 1.5 h; 5, ATP + GTP + DncV/RNase T1 at 37°C for 1.5 h; 6, ATP + GTP + DncV/RNase T2 at 37°C for 1.5 h. (B) 1, ATP + GTP trinucleotide control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/S1 nuclease; 4, c-di-GMP + S1 nuclease; 5, c-di-AMP + S1 nuclease. (C) 1, ATP + GTP; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/RNase T2; 4, c-di-GMP + RNase T2; 5, c-di-AMP + RNase T2. (D) Expected enzymatic products of cGAMP isomers upon digestion with the RNase enzymes RNase T1 (T1), S1 nuclease (S1), and RNase T2 (T2). pG = 5′-GMP, Gp = 3′-GMP, pA = 5′-AMP, Ap = 3′-AMP.
    Figure Legend Snippet: Structural characterization of the DncV reaction product demonstrated a 3′ 3′-linked cGAMP. Digestion of DncV-produced cGAMP with S1 nuclease, RNase T1, and RNase T2 was performed. DncV (5 µM) was incubated with 5 mM ATP + GTP, ATP, or GTP for 1.5 h at 37°C as indicated under A–C. Subsequently, 1 µl of RNase T1, S1 nuclease, or RNase T2 was added for 1 h at 37°C. Products were run on a TLC plate for 30 min at room temperature. (A) 1, ATP + GTP nucleoside triphosphate substrate control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV at 37°C for 1.5 h; 4, ATP + GTP + DncV/S1 nuclease at 37°C for 1.5 h; 5, ATP + GTP + DncV/RNase T1 at 37°C for 1.5 h; 6, ATP + GTP + DncV/RNase T2 at 37°C for 1.5 h. (B) 1, ATP + GTP trinucleotide control; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/S1 nuclease; 4, c-di-GMP + S1 nuclease; 5, c-di-AMP + S1 nuclease. (C) 1, ATP + GTP; 2, ATP + GTP + DncV; 3, ATP + GTP + DncV/RNase T2; 4, c-di-GMP + RNase T2; 5, c-di-AMP + RNase T2. (D) Expected enzymatic products of cGAMP isomers upon digestion with the RNase enzymes RNase T1 (T1), S1 nuclease (S1), and RNase T2 (T2). pG = 5′-GMP, Gp = 3′-GMP, pA = 5′-AMP, Ap = 3′-AMP.

    Techniques Used: Produced, Incubation, Thin Layer Chromatography

    2) Product Images from "Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation"

    Article Title: Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx097

    CRISPR interference leads to appearance of S1 nuclease sensitive target DNA  in vivo . ( A ) Three  E. coli  strains used are shown. KD263 is capable of both CRISPR interference and adaptation. KD454 is incapable of CRISPR interference. BW40297 is capable of interference but not adaptation. ( B ) Total DNA was prepared from cells shown in A transformed with pG8mut and grown in the presence or in the absence of  cas  gene expression inducers. Half of each sample was treated with S1 nuclease. qPCR with primer pairs amplifying a 210 nucleotide plasmid fragment or 138 nucleotide CRISPR leader fragment was performed. For each sample, Ct values are presented. For KD263 and BW40297 samples, carrying pG8mut plasmid, higher Ct values observed for plasmid amplicon in the presence of inducers are due to CRISPR interference.
    Figure Legend Snippet: CRISPR interference leads to appearance of S1 nuclease sensitive target DNA in vivo . ( A ) Three E. coli strains used are shown. KD263 is capable of both CRISPR interference and adaptation. KD454 is incapable of CRISPR interference. BW40297 is capable of interference but not adaptation. ( B ) Total DNA was prepared from cells shown in A transformed with pG8mut and grown in the presence or in the absence of cas gene expression inducers. Half of each sample was treated with S1 nuclease. qPCR with primer pairs amplifying a 210 nucleotide plasmid fragment or 138 nucleotide CRISPR leader fragment was performed. For each sample, Ct values are presented. For KD263 and BW40297 samples, carrying pG8mut plasmid, higher Ct values observed for plasmid amplicon in the presence of inducers are due to CRISPR interference.

    Techniques Used: CRISPR, In Vivo, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    3) Product Images from "Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects"

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108401

    Illustration of the label-free ssDNA detection strategy for detecting sequence-specific ssDNA target with its complementary ssDNA, S1 nuclease and DNA fluorescent dyes.
    Figure Legend Snippet: Illustration of the label-free ssDNA detection strategy for detecting sequence-specific ssDNA target with its complementary ssDNA, S1 nuclease and DNA fluorescent dyes.

    Techniques Used: Sequencing

    Detection results of Sequence-specificity ssDNA in different surroundings. (A). Fluorescence intensities of three viruses ssDNA and their hybridized products after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. a, H1N1-dsDNA; b, CaM-dsDNA; c, HCV-dsDNA; d, H1N1-ssDNA; e, CaM-ssDNA; f, HCV-ssDNA; g, blank. (B). Fluorescence intensities of three viruses ssDNA and their hybridized products in 4% pork muscle tissue homogenate after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. (C). Fluorescence intensities of 50 pM HCV-ssDNA and its hybridized products in 0.5 µg·mL −1  Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×). (D). Fluorescence intensities of 50 nM HCV-ssDNA and its hybridized products in 5 µg·mL −1  Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×).
    Figure Legend Snippet: Detection results of Sequence-specificity ssDNA in different surroundings. (A). Fluorescence intensities of three viruses ssDNA and their hybridized products after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. a, H1N1-dsDNA; b, CaM-dsDNA; c, HCV-dsDNA; d, H1N1-ssDNA; e, CaM-ssDNA; f, HCV-ssDNA; g, blank. (B). Fluorescence intensities of three viruses ssDNA and their hybridized products in 4% pork muscle tissue homogenate after S1 nuclease-mediated cleavage using SYBR Gold (1×). The concentration of target ssDNAs are 50 nM, and their complementary ssDNAs are 100 nM. (C). Fluorescence intensities of 50 pM HCV-ssDNA and its hybridized products in 0.5 µg·mL −1 Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×). (D). Fluorescence intensities of 50 nM HCV-ssDNA and its hybridized products in 5 µg·mL −1 Lambda DNA standard solution after S1 nuclease-mediated cleavage using SYBR Gold (1×).

    Techniques Used: Sequencing, Fluorescence, Concentration Assay, Chick Chorioallantoic Membrane Assay, Lambda DNA Preparation

    Fluorescence intensity of oligonucleotides hybridized products with 1× PicoGreen after S1 nuclease-mediated cleavage. The concentration of oligonucleotides and their complementary oligonucleotides is 50 nM and 100 nM respectively. (A) The effects of lengths of target ssDNA on fluorescence intensity. (B) The effects of nucleotide composition of target ssDNA on fluorescence intensity. (C) Linearity of the proposed method (three virus ssDNA). Plots of fluorescence intensity versus different concentrations of target ssDNA hybridized products (linear range, 50 pM–64 nM).
    Figure Legend Snippet: Fluorescence intensity of oligonucleotides hybridized products with 1× PicoGreen after S1 nuclease-mediated cleavage. The concentration of oligonucleotides and their complementary oligonucleotides is 50 nM and 100 nM respectively. (A) The effects of lengths of target ssDNA on fluorescence intensity. (B) The effects of nucleotide composition of target ssDNA on fluorescence intensity. (C) Linearity of the proposed method (three virus ssDNA). Plots of fluorescence intensity versus different concentrations of target ssDNA hybridized products (linear range, 50 pM–64 nM).

    Techniques Used: Fluorescence, Concentration Assay

    F/F0 values of three viruses and three types of dyes obtained from the proposed strategy. The concentrations of target ssDNA and their complementary ssDNAs are 50 nM and 100 nM respectively, and the concentration of dyes is 1×. F0 represents the fluorescent intensity of solution blank containing S1 nuclease, HB solution and DNA dyes, and F represents the fluorescent intensity of the solution as described above upon addition of target ssDNA, or target ssDNA and its complementary sequence.
    Figure Legend Snippet: F/F0 values of three viruses and three types of dyes obtained from the proposed strategy. The concentrations of target ssDNA and their complementary ssDNAs are 50 nM and 100 nM respectively, and the concentration of dyes is 1×. F0 represents the fluorescent intensity of solution blank containing S1 nuclease, HB solution and DNA dyes, and F represents the fluorescent intensity of the solution as described above upon addition of target ssDNA, or target ssDNA and its complementary sequence.

    Techniques Used: Concentration Assay, Sequencing

    4) Product Images from "nextPARS: parallel probing of RNA structures in Illumina"

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    Journal:

    doi: 10.1261/rna.063073.117

    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A)+ RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A)+ RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).
    Figure Legend Snippet: Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A)+ RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A)+ RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Techniques Used: In Vitro, Sample Prep, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Sequencing

    5) Product Images from "Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications"

    Article Title: Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

    Journal:

    doi: 10.1110/ps.062082606

    Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.
    Figure Legend Snippet: Gene fragmentation. ( A ) Schematic of the CDH gene fragmentation process. PCR with TTP/dUTP mixtures is used to generate copies of the target gene in which uracil is randomly incorporated in place of thymine. The uracil-doped amplified DNA is subjected to a modified base-excision cascade in which uracil-DNA glycosylase excises the uracil bases generating abasic sites, which are cleaved by endonuclease IV, giving a single-strand nick that is converted to a double-strand break and blunt-ended by S1 nuclease. As the reaction cascade is initiated only at uracils, whose distribution along the sequence and among the PCR reaction products is random, the cascade generates a random and unbiased library of gene fragments, whose size distribution is solely dictated by the TTP/dUTP ratio. ( B ) dUTP-dose dependent fragmentation. SYBR-Safe stained 1% agarose gel of an ∼2.2-kb human p85α PCR-amplified cDNA ( right- hand lane), alongside the products of CDH fragmentation reactions using increasing amounts of dUTP (as percent of total TTP+dUTP concentration). The progressive decrease in modal size of the DNA distribution with increasing dUTP concentration is clearly seen.

    Techniques Used: Polymerase Chain Reaction, Amplification, Modification, Sequencing, Staining, Agarose Gel Electrophoresis, Concentration Assay

    6) Product Images from "Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array"

    Article Title: Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-7-251

    Array analysis of symmetrical and asymmetrical PCR products. A.  Signal intensity for hybridisation of symmetrical and asymmetrical PCR products of 312 and 317 bases, respectively, both with a 5′ segment length of 43 bases. Asymmetrical PCR was carried out using a forward primer modified for linear-after-the-exponential (LATE) PCR, with a primer ratio of 1:10. Approximately 500 ng total DNA (single-stranded plus double-stranded) was hybridised to each array. Results shown are mean values for duplicate spots on the same array. AA = AA mismatch between probe and PCR product, etc.  B.  Agarose gel electrophoresis of symmetrical and asymmetrical PCR products. The products of symmetrical and asymmetrical PCR were visualised by agarose gel electrophoresis with GelRed nucleic acid stain before and after treatment with S1 nuclease to confirm that the product of asymmetrical PCR contained single-stranded DNA (removed by S1 nuclease) as well as double-stranded DNA.
    Figure Legend Snippet: Array analysis of symmetrical and asymmetrical PCR products. A. Signal intensity for hybridisation of symmetrical and asymmetrical PCR products of 312 and 317 bases, respectively, both with a 5′ segment length of 43 bases. Asymmetrical PCR was carried out using a forward primer modified for linear-after-the-exponential (LATE) PCR, with a primer ratio of 1:10. Approximately 500 ng total DNA (single-stranded plus double-stranded) was hybridised to each array. Results shown are mean values for duplicate spots on the same array. AA = AA mismatch between probe and PCR product, etc. B. Agarose gel electrophoresis of symmetrical and asymmetrical PCR products. The products of symmetrical and asymmetrical PCR were visualised by agarose gel electrophoresis with GelRed nucleic acid stain before and after treatment with S1 nuclease to confirm that the product of asymmetrical PCR contained single-stranded DNA (removed by S1 nuclease) as well as double-stranded DNA.

    Techniques Used: Polymerase Chain Reaction, Hybridization, Modification, Agarose Gel Electrophoresis, Electrophoresis, Staining

    7) Product Images from "RNA secondary structure profiling in zebrafish reveals unique regulatory features"

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4497-0

    Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
    Figure Legend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

    Techniques Used: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

    Comparison of RNA structures of  ubc  3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease.  a . Bar plot represents PARS scores of 3’UTR region of  ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS.  b . Enzymatic footprinting of  ubc  3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions.  c . Heatmap representing secondary structure of 68 positions of  ubc  3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions
    Figure Legend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

    Techniques Used: Footprinting

    8) Product Images from "Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress"

    Article Title: Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.08.065

    FANCJ Contributes to Unrestrained Replication and S1 Nuclease Sensitivity in HLTF-Depleted Cells (A) Western blot analysis with the indicated Abs of lysates from HCT116 cells expressing shRNA against FANCJ, HLTF, or NSC. (B) Schematic, representative images, and quantification of CldU tract length during HU treatment. (C) Schematic and quantification of the CldU/IdU ratio in the indicated shRNA-expressing HCT116 cells with or without S1 nuclease incubation. Each dot represents one fiber; at least 200 fibers are quantified from two independent experiments. Red bars represent the median. Statistical analysis according to two-tailed Mann-Whitney test; ****p
    Figure Legend Snippet: FANCJ Contributes to Unrestrained Replication and S1 Nuclease Sensitivity in HLTF-Depleted Cells (A) Western blot analysis with the indicated Abs of lysates from HCT116 cells expressing shRNA against FANCJ, HLTF, or NSC. (B) Schematic, representative images, and quantification of CldU tract length during HU treatment. (C) Schematic and quantification of the CldU/IdU ratio in the indicated shRNA-expressing HCT116 cells with or without S1 nuclease incubation. Each dot represents one fiber; at least 200 fibers are quantified from two independent experiments. Red bars represent the median. Statistical analysis according to two-tailed Mann-Whitney test; ****p

    Techniques Used: Western Blot, Expressing, shRNA, Incubation, Two Tailed Test, MANN-WHITNEY

    9) Product Images from "Electrical discharges in water induce spores’ DNA damage"

    Article Title: Electrical discharges in water induce spores’ DNA damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201448

    Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a)  Pulsed-Field Gel electrophoresis (PFGE) patterns of  NotI -digested chromosomal DNA (double-strand) from  Bacillus pumilus  spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12).  (b)  Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.
    Figure Legend Snippet: Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a) Pulsed-Field Gel electrophoresis (PFGE) patterns of NotI -digested chromosomal DNA (double-strand) from Bacillus pumilus spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12). (b) Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Migration, Fluorescence

    10) Product Images from "Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan"

    Article Title: Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan

    Journal:

    doi: 10.1128/AAC.03242-14

    (a) Pulsed-field gel of S1 nuclease-digested genomic DNA from  A. bereziniae  CHI-40-1, recipients, and transconjugants; (b) in-gel hybridization with a  bla NDM-1  gene probe; (c) pulsed-field gel of ApaI-digested genomic DNA from CHI-40-1; (d) in-gel hybridization
    Figure Legend Snippet: (a) Pulsed-field gel of S1 nuclease-digested genomic DNA from A. bereziniae CHI-40-1, recipients, and transconjugants; (b) in-gel hybridization with a bla NDM-1 gene probe; (c) pulsed-field gel of ApaI-digested genomic DNA from CHI-40-1; (d) in-gel hybridization

    Techniques Used: Pulsed-Field Gel, Hybridization

    11) Product Images from "Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy"

    Article Title: Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

    Journal: Scientific Reports

    doi: 10.1038/srep03759

    Binding and infection specificity of rAAV-2 particles for different cell types. (a) 1.8 × 10 9  viral particles were incubated on fixed 2.0 × 10 3  A431 or HeLa cells, respectively. Unbound particles were removed by washing and intact capsids were detected via A20 antibody. All values are expressed relative to binding of AAV-2 ΔHSPG  to HeLa cells, which was set to 100% (n = 5, mean ± SD). (b) 10 4  A431, HeLa, or MCF7 cells were incubated with 3 × 10 8  genomic viral particles for 24 h. Subsequently, the cells were harvested, digested with proteinase K and incubated with S1 nuclease. Double-stranded viral DNA was then quantified using qPCR (n = 3, mean ± SD).
    Figure Legend Snippet: Binding and infection specificity of rAAV-2 particles for different cell types. (a) 1.8 × 10 9 viral particles were incubated on fixed 2.0 × 10 3 A431 or HeLa cells, respectively. Unbound particles were removed by washing and intact capsids were detected via A20 antibody. All values are expressed relative to binding of AAV-2 ΔHSPG to HeLa cells, which was set to 100% (n = 5, mean ± SD). (b) 10 4 A431, HeLa, or MCF7 cells were incubated with 3 × 10 8 genomic viral particles for 24 h. Subsequently, the cells were harvested, digested with proteinase K and incubated with S1 nuclease. Double-stranded viral DNA was then quantified using qPCR (n = 3, mean ± SD).

    Techniques Used: Binding Assay, Infection, Incubation, Real-time Polymerase Chain Reaction

    12) Product Images from "The p53 Tumor Suppressor Protein Does Not Regulate Expression of Its Own Inhibitor, MDM2, Except under Conditions of Stress"

    Article Title: The p53 Tumor Suppressor Protein Does Not Regulate Expression of Its Own Inhibitor, MDM2, Except under Conditions of Stress

    Journal:

    doi:

    RNAs from both the P1 and P2 promoters of mdm2 are expressed in murine tissues. (A) Schematic of exons 1 to 3 of the mdm2 gene and of the S1 nuclease protection assay used to distinguish mdm2 transcripts from the P1 and P2 promoters. Indicated are the predicted sizes of hybridization products following digestion with S1 nuclease. (B) The amounts of mdm2 RNA from the P1 and P2 promoters were compared in spleen (lanes 1 to 3), thymus (lanes 4 to 6), kidney (lanes 7 to 9), heart (lanes 10 to 12), brain (lanes 13 to 15), and liver (lanes 16 to 18). The indicated tissues were isolated from three age-matched wild-type FVB/N animals. Twenty-five micrograms of total RNA from each tissue was analyzed for mdm2 RNA from the P1 and P2 promoters by S1 nuclease digestion following hybridization to a radiolabeled, denatured DNA probe complementary to mdm2 exons 1 to 3. The band that appears sporadically in some samples is an artifact from the probe.
    Figure Legend Snippet: RNAs from both the P1 and P2 promoters of mdm2 are expressed in murine tissues. (A) Schematic of exons 1 to 3 of the mdm2 gene and of the S1 nuclease protection assay used to distinguish mdm2 transcripts from the P1 and P2 promoters. Indicated are the predicted sizes of hybridization products following digestion with S1 nuclease. (B) The amounts of mdm2 RNA from the P1 and P2 promoters were compared in spleen (lanes 1 to 3), thymus (lanes 4 to 6), kidney (lanes 7 to 9), heart (lanes 10 to 12), brain (lanes 13 to 15), and liver (lanes 16 to 18). The indicated tissues were isolated from three age-matched wild-type FVB/N animals. Twenty-five micrograms of total RNA from each tissue was analyzed for mdm2 RNA from the P1 and P2 promoters by S1 nuclease digestion following hybridization to a radiolabeled, denatured DNA probe complementary to mdm2 exons 1 to 3. The band that appears sporadically in some samples is an artifact from the probe.

    Techniques Used: Hybridization, Isolation

    13) Product Images from "Electrical discharges in water induce spores’ DNA damage"

    Article Title: Electrical discharges in water induce spores’ DNA damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201448

    Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a)  Pulsed-Field Gel electrophoresis (PFGE) patterns of  NotI -digested chromosomal DNA (double-strand) from  Bacillus pumilus  spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12).  (b)  Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.
    Figure Legend Snippet: Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a) Pulsed-Field Gel electrophoresis (PFGE) patterns of NotI -digested chromosomal DNA (double-strand) from Bacillus pumilus spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12). (b) Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Migration, Fluorescence

    14) Product Images from "Effect of Neisseria meningitidis Fur Mutations on Global Control of Gene Transcription"

    Article Title: Effect of Neisseria meningitidis Fur Mutations on Global Control of Gene Transcription

    Journal:

    doi: 10.1128/JB.188.7.2483-2492.2006

    (A) Upregulation of σ 32 promoters in the MC-Fko fur -null mutant. An S1 nuclease assay on DNA probes corresponding to the dnaK , lon , clpB , and groE heat shock genes was conducted as described in Materials and Methods. Total RNA from MC58 (wt),
    Figure Legend Snippet: (A) Upregulation of σ 32 promoters in the MC-Fko fur -null mutant. An S1 nuclease assay on DNA probes corresponding to the dnaK , lon , clpB , and groE heat shock genes was conducted as described in Materials and Methods. Total RNA from MC58 (wt),

    Techniques Used: Mutagenesis, Nuclease Assay

    15) Product Images from "RNA secondary structure profiling in zebrafish reveals unique regulatory features"

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4497-0

    Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
    Figure Legend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

    Techniques Used: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

    Comparison of RNA structures of  ubc  3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease.  a . Bar plot represents PARS scores of 3’UTR region of  ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS.  b . Enzymatic footprinting of  ubc  3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions.  c . Heatmap representing secondary structure of 68 positions of  ubc  3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions
    Figure Legend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

    Techniques Used: Footprinting

    16) Product Images from "Distribution of Plasmids in Distinct Leptospira Pathogenic Species"

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0004220

    Identification of homologous sequence of lcp1- rep , lcp2- rep  and lcp3- rep  in 15 Chinese epidemic  Leptospira  strains. Leptospira  strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions, blotted to a nylon membrane and then hybridized with the probes of lcp1- rep (A), lcp2- rep (B) and lcp3- rep (C). Probes were generated by PCR of T-vector plasmid DNA containing  rep  genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively (  S2 Table ). Markers are shown on the right. 56601–56655 represent 15  Leptospira  Chinese epidemic strains as detailed in   Table 3 .
    Figure Legend Snippet: Identification of homologous sequence of lcp1- rep , lcp2- rep and lcp3- rep in 15 Chinese epidemic Leptospira strains. Leptospira strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions, blotted to a nylon membrane and then hybridized with the probes of lcp1- rep (A), lcp2- rep (B) and lcp3- rep (C). Probes were generated by PCR of T-vector plasmid DNA containing rep genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively ( S2 Table ). Markers are shown on the right. 56601–56655 represent 15 Leptospira Chinese epidemic strains as detailed in Table 3 .

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Plasmid Preparation

    Detection of lbp1 and lbp2 plasmids by PFGE. ( A) S1-PFGE-based Southern blot analysis. (B) Single restriction enzyme digestion of the plasmids followed by Southern blot analysis. M represents the standard strain  Salmonella enterica  serotype Braenderup (H9812) digested with  Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. ND, undigested  L .  borgpetersenii  serovar Ballum strain 56604;  L .  borgpetersenii  serovar Ballum strain 56604 were digested by S1 nuclease,  Not I and  Pst I. For Southern blot analysis, the genomic DNA of  L .  borgpetersenii  serovar Ballum strain 56604 was blotted to a nylon membrane and hybridized by lcp1- rep  and lcp2- rep . Probes were generated by PCR of T-vector plasmid DNA containing  rep  genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively (  S2 Table ).
    Figure Legend Snippet: Detection of lbp1 and lbp2 plasmids by PFGE. ( A) S1-PFGE-based Southern blot analysis. (B) Single restriction enzyme digestion of the plasmids followed by Southern blot analysis. M represents the standard strain Salmonella enterica serotype Braenderup (H9812) digested with Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. ND, undigested L . borgpetersenii serovar Ballum strain 56604; L . borgpetersenii serovar Ballum strain 56604 were digested by S1 nuclease, Not I and Pst I. For Southern blot analysis, the genomic DNA of L . borgpetersenii serovar Ballum strain 56604 was blotted to a nylon membrane and hybridized by lcp1- rep and lcp2- rep . Probes were generated by PCR of T-vector plasmid DNA containing rep genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively ( S2 Table ).

    Techniques Used: Southern Blot, Molecular Weight, Marker, Generated, Polymerase Chain Reaction, Plasmid Preparation

    Plasmids detection in 15 Chinese epidemic  Leptospira  strains by S1-PFGE separation. Leptospira  strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions. Marker represents the pattern of the standard strain  Salmonella enterica  serotype Braenderup (H9812) digested with  Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. M represents the marker electrophoresed with each sample. 56601–56655 represent 15 Chinese epidemic  Leptospira  strains as detailed in   Table 3 .
    Figure Legend Snippet: Plasmids detection in 15 Chinese epidemic Leptospira strains by S1-PFGE separation. Leptospira strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions. Marker represents the pattern of the standard strain Salmonella enterica serotype Braenderup (H9812) digested with Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. M represents the marker electrophoresed with each sample. 56601–56655 represent 15 Chinese epidemic Leptospira strains as detailed in Table 3 .

    Techniques Used: Marker, Molecular Weight

    17) Product Images from "Electrical discharges in water induce spores’ DNA damage"

    Article Title: Electrical discharges in water induce spores’ DNA damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201448

    Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a)  Pulsed-Field Gel electrophoresis (PFGE) patterns of  NotI -digested chromosomal DNA (double-strand) from  Bacillus pumilus  spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12).  (b)  Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.
    Figure Legend Snippet: Electric arcs induce single-strand DNA breaks, base excisions and pyrimidines dimers. (a) Pulsed-Field Gel electrophoresis (PFGE) patterns of NotI -digested chromosomal DNA (double-strand) from Bacillus pumilus spores exposed (+) or not (-) to electric arcs. The DNA migration was observed with (+) and without (-) various endonuclease enzymes. The S1 nuclease cleaves the single-strand DNA; the endonuclease IV cleaves the apurinic/apyrimidinic sites; the endonuclease V cleaves the pyrimidine dimers. The size range of the standard Lambda Ladder is between 48.5 and 1000 kb. In absence of electric arcs (lanes 1, 2, 5, 6, 9, 11), DNA was intact. After electric arcs exposure; the migration of fragments was slowed down (lane 3); the S1 nuclease revealed single-strand DNA breaks (lane 4); the combination with the endonucleases S1 and IV exhibited base excisions (lane 8); the combination with the endonucleases S1 and V showed the presence of pyrimidine dimers (lane 12). (b) Example of quantification of the fluorescence intensity (in percentage compared to the control, lane 1) on the higher fragment (between 242.5 and 291 kb), calculated from 3 independent experiments. Lane 1: control; Lane 3: loss of intensity of 15% with electric arcs alone; Lane 4: loss of 75% with electric arcs and S1 nuclease; Lane 8: loss of 85% with electric arcs and S1 nuclease combined with endonuclease IV; Lane 12: loss of 93% with electric arcs and S1 nuclease combined with endonuclease V.

    Techniques Used: Pulsed-Field Gel, Electrophoresis, Migration, Fluorescence

    18) Product Images from "RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events"

    Article Title: RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm447

    In vitro  transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases ( A : lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled  32 P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold ( B ).
    Figure Legend Snippet: In vitro transcribed ATM Δ RNA enzymatically digested with S1 nuclease, T1 and V1 RNases ( A : lanes S1, T1 and V1). No enzyme was added to the RNA in a control reaction mixture (A: lane C). The RNA substrate on which this analysis was perfomed consisted of the entire RNA transcript from the ATM Δ RNA plasmid (∼700 nt). By providing an extensive background of flanking RNA molecule we have aimed to minimize any folding bias that may have derived from analysing the pseudoexon sequence alone. The cleaved fragments were detected by performing a RT reaction using a labelled 32 P-end labelled antisense oligo and separating them in a denaturing 6% polyacrylamide gel. A sequencing reaction performed with the same RT primer was run in parallel to the cleavages in order to determine the cleavage sites (A: lanes G, A, T, C). Squares, circles and triangles indicate S1, T1 and V1 cleavage sites. Black and white symbols indicate high and low cleavage intensity, respectively. RT stops are indicated by arrows. The positions of the 3′pe, 5′int and 5′pe splice sites are indicated on the right. The observed cleavages were then compared with the ATM folding predictions by mFold ( B ).

    Techniques Used: In Vitro, Plasmid Preparation, Derivative Assay, Sequencing

    19) Product Images from "Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes"

    Article Title: Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03675-1

    Measuring RNA-ligand binding by structure probing and deep sequencing.  a  RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes).  b  Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding.  c  PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in  B .  subtilis  and  P .  aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively.  d  PARCEL sequencing data for the  B .  subtilis  TPP riboswitch, thiT, in the presence and absence of 100 µM TPP (top), 100 µM thiamine (middle), and 100 µM oxythiamine (bottom). PARCEL detected strongest structural change in thiT in the presence of TPP, followed by thiamine and then oxythiamine, which corresponds to the binding affinities of TPP riboswitches for these metabolites  9 .  e  The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the  B .  subtilis  transcriptome
    Figure Legend Snippet: Measuring RNA-ligand binding by structure probing and deep sequencing. a RNA undergoes structure changes upon ligand binding. This structural change is detected by the double-strand specific nuclease, RNase V1, which cuts at different double-stranded places along the RNA in the presence and absence of the ligand. The cleavage sites are then captured and cloned into a cDNA library for deep sequencing. After mapping the reads to the transcriptome, we can identify which bases have undergone changes in structuredness upon ligand binding (highlighted in beige boxes). b Deep sequencing reveals structure changes of a known TPP riboswitch, thiM, using RNase V1 (top), S1 nuclease (middle), and in-line probing (bottom). The red and black lines indicate the structure profiles of thiM treated with and without 100 µM TPP, respectively. The beige regions highlight regions of structural changes upon ligand binding. c PARCEL identified 85% of known TPP, FMN, and SAM riboswitches in B . subtilis and P . aeruginosa . The black and the white bars indicate the number of known riboswitches that were captured and missed in our study, respectively. d PARCEL sequencing data for the B . subtilis TPP riboswitch, thiT, in the presence and absence of 100 µM TPP (top), 100 µM thiamine (middle), and 100 µM oxythiamine (bottom). PARCEL detected strongest structural change in thiT in the presence of TPP, followed by thiamine and then oxythiamine, which corresponds to the binding affinities of TPP riboswitches for these metabolites 9 . e The plots show normalized V1 read counts of the thiC TPP riboswitch under increasing concentrations of TPP. PARCEL was performed on the B . subtilis transcriptome

    Techniques Used: Ligand Binding Assay, Sequencing, Clone Assay, cDNA Library Assay, Binding Assay

    20) Product Images from "The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis"

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis

    Journal:

    doi: 10.1128/JB.00849-08

    (A) Diagrammatic representation of the sdhCDAB locus and the extended region of complementarity found between the NrrF and the sdhDA mRNA gene junction. The relative positions of the radioactively labeled probes designed for the S1 nuclease assay analysis are shown. (B) The predicted structure (Mfold) of the NrrF sRNA resulting in a free energy (Δ G °r ) of −42.2. The 5′ end was identified by primer extension, and the 3′ end was deduced from the prediction of the rho-independent terminator within the sequence. The predicted AU-rich Hfq binding site between 94 and 104 nt in the sequence is boxed, and the nucleotides involved in the putative base-pairing with sdhDA mRNA are shown in boldface. The arrows indicate the positions of the first and last nucleotides of the deletion of the loop that was generated for the NrrfΔ31-58 mutant transcript (see Fig. ).
    Figure Legend Snippet: (A) Diagrammatic representation of the sdhCDAB locus and the extended region of complementarity found between the NrrF and the sdhDA mRNA gene junction. The relative positions of the radioactively labeled probes designed for the S1 nuclease assay analysis are shown. (B) The predicted structure (Mfold) of the NrrF sRNA resulting in a free energy (Δ G °r ) of −42.2. The 5′ end was identified by primer extension, and the 3′ end was deduced from the prediction of the rho-independent terminator within the sequence. The predicted AU-rich Hfq binding site between 94 and 104 nt in the sequence is boxed, and the nucleotides involved in the putative base-pairing with sdhDA mRNA are shown in boldface. The arrows indicate the positions of the first and last nucleotides of the deletion of the loop that was generated for the NrrfΔ31-58 mutant transcript (see Fig. ).

    Techniques Used: Labeling, Nuclease Assay, Sequencing, Binding Assay, Generated, Mutagenesis

    (A) Diagrammatic representation of the locus containing the nrrF gene in MC58. The Fur-regulated promoter is indicated in gray, the orientation of the sRNA is indicated with a black arrow, and the relative position of the rho-independent transcriptional terminator is marked with a hairpin loop. (B) Mapping of the 5′ end of the nrrF transcript by primer extension. Portions (20 μg) of total RNA prepared from cultures of the wild type (MC58) and Fur-null mutant (MC-Fko) grown to mid-logarithmic phase under iron-replete conditions were hybridized with the sR-p7 primer (Table ) and elongated with reverse transcriptase. The elongated primer band mapping the 5′ end of the sRNA transcript is indicated. Sequence reactions (G, A, T, and C) were performed with the same primer on plasmid pGemsRNA1/2 as a template. The corresponding +1 nucleotide of transcriptional initiation and the upstream promoter sequences are indicated on the left. (C) DNase I footprinting analysis with purified meningococcal Fur protein and a radioactively labeled 245-bp DNA probe, 5′ end labeled at the EcoRI site, corresponding to the nrrF promoter region. The probe was incubated with increasing concentrations of Fur protein: lanes 1 to 6 correspond to concentrations of 0, 14 nM, 44 nM, 130 nM, 392 nM, and 1.2 μM concentrations of Fur protein. A G+A sequencing reaction of the probe was performed and run in parallel as a molecular weight ladder. The box and arrow to the left show the position and the direction of the Fur-box and nrrF gene, respectively. The Fur-protected region is indicated to the right as a vertical black bar, and the numbers indicate the boundaries of the binding site with respect to the +1 transcriptional initiation site. (D) Regulation of NrrF transcription. Total RNA was prepared from the wild type (MC58), the Fur-null mutant (MC-Fko), its complemented derivative (MC-Fko-C), the Hfq mutant (Δ hfq ), and the Fur and Hfq double mutant (Fko-Δ hfq ) grown to mid-log phase under iron-replete conditions before (+) and after (−) 15 min of treatment with iron chelator (2,2′-dipyridyl). Then, 10 μg of RNA from each strain was reverse transcribed with the sR-p7 primer, and the relative quantities of extended primer product are shown from a single representative experiment. (E) Time course experiment in which cultures of MC58 and MC-Fko strains were grown in iron-replete conditions and total RNA was extracted after 1, 2, and 3 h (logarithmic phase) and 5 and 7 h (stationary phase). The relative quantities of NrrF and tbp2 transcripts were analyzed from 10 μg of each total RNA sample by quantitative primer extension and S1 nuclease assay, respectively.
    Figure Legend Snippet: (A) Diagrammatic representation of the locus containing the nrrF gene in MC58. The Fur-regulated promoter is indicated in gray, the orientation of the sRNA is indicated with a black arrow, and the relative position of the rho-independent transcriptional terminator is marked with a hairpin loop. (B) Mapping of the 5′ end of the nrrF transcript by primer extension. Portions (20 μg) of total RNA prepared from cultures of the wild type (MC58) and Fur-null mutant (MC-Fko) grown to mid-logarithmic phase under iron-replete conditions were hybridized with the sR-p7 primer (Table ) and elongated with reverse transcriptase. The elongated primer band mapping the 5′ end of the sRNA transcript is indicated. Sequence reactions (G, A, T, and C) were performed with the same primer on plasmid pGemsRNA1/2 as a template. The corresponding +1 nucleotide of transcriptional initiation and the upstream promoter sequences are indicated on the left. (C) DNase I footprinting analysis with purified meningococcal Fur protein and a radioactively labeled 245-bp DNA probe, 5′ end labeled at the EcoRI site, corresponding to the nrrF promoter region. The probe was incubated with increasing concentrations of Fur protein: lanes 1 to 6 correspond to concentrations of 0, 14 nM, 44 nM, 130 nM, 392 nM, and 1.2 μM concentrations of Fur protein. A G+A sequencing reaction of the probe was performed and run in parallel as a molecular weight ladder. The box and arrow to the left show the position and the direction of the Fur-box and nrrF gene, respectively. The Fur-protected region is indicated to the right as a vertical black bar, and the numbers indicate the boundaries of the binding site with respect to the +1 transcriptional initiation site. (D) Regulation of NrrF transcription. Total RNA was prepared from the wild type (MC58), the Fur-null mutant (MC-Fko), its complemented derivative (MC-Fko-C), the Hfq mutant (Δ hfq ), and the Fur and Hfq double mutant (Fko-Δ hfq ) grown to mid-log phase under iron-replete conditions before (+) and after (−) 15 min of treatment with iron chelator (2,2′-dipyridyl). Then, 10 μg of RNA from each strain was reverse transcribed with the sR-p7 primer, and the relative quantities of extended primer product are shown from a single representative experiment. (E) Time course experiment in which cultures of MC58 and MC-Fko strains were grown in iron-replete conditions and total RNA was extracted after 1, 2, and 3 h (logarithmic phase) and 5 and 7 h (stationary phase). The relative quantities of NrrF and tbp2 transcripts were analyzed from 10 μg of each total RNA sample by quantitative primer extension and S1 nuclease assay, respectively.

    Techniques Used: Mutagenesis, Sequencing, Plasmid Preparation, Footprinting, Purification, Labeling, Incubation, Molecular Weight, Binding Assay, Nuclease Assay

    Related Articles

    Centrifugation:

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA
    Article Snippet: After centrifugation at 16,000 × g max for 30 min at 4 °C, the DNA pellet was dissolved overnight in TE buffer (10 mm Tris/HCl and 1 mm EDTA, pH 7.8) at 55 °C. mtDNA was quantified by qPCR using primers against ND5 with RpL32 as an internal standard (see Table S1 ) in a StepOnePlus Real-Time PCR System with a Fast SYBR Green Master Mix kit. .. For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min.

    Amplification:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: The sodB probe, a 292-bp fragment, was amplified by PCR with Sod1 and Sod2 primers (Table ), end labeled, digested with EcoRI, and purified by using Chromaspin TE-100 columns. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Synthesized:

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    Lambda DNA Preparation:

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: The fluorescent dyes SYBR Green II (10,000× concentrated), SYBR Gold (10,000× concentrated), and PicoGreen dsDNA Reagent and Kits (200× concentrated, including 100 µg·mL−1 Lambda DNA standard) were purchased from Invitrogen (CA, USA). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    Real-time Polymerase Chain Reaction:

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA
    Article Snippet: After centrifugation at 16,000 × g max for 30 min at 4 °C, the DNA pellet was dissolved overnight in TE buffer (10 mm Tris/HCl and 1 mm EDTA, pH 7.8) at 55 °C. mtDNA was quantified by qPCR using primers against ND5 with RpL32 as an internal standard (see Table S1 ) in a StepOnePlus Real-Time PCR System with a Fast SYBR Green Master Mix kit. .. For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min.

    Incubation:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation.

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA
    Article Snippet: To analyze mtDNA topology, integrity, and replication intermediates, mitochondrial nucleic acid was prepared from S2 cells, adult flies, and L3 larvae as described previously ( ). .. For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min. .. Reactions were deproteinized by phenol-chloroform extraction, and 2 μl of the aqueous phase was analyzed by 0.5% agarose gel electrophoresis (30-cm gels run for 40 h at 1.7 V/cm in Tris/Borate/EDTA).

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: For experiments with cells expressing photolyases, upon UV irradiation, the cells were incubated with 200 μM IdU in PBS+ supplemented with 5% FBS for 60 min at RT on the photoreactivation apparatus. .. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC. .. See Supplementary Material for more details.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: The FFPE genome library construction protocol and sequencing steps were as described previously ( ) with some modifications. .. S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Significance Assay:

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: The libraries were sequenced on Ion Proton platform (Life Technologies, CA, US) employing semiconductor based chemistry after quality check to generate single end reads. .. Sequencing was done for five technical replicates each for RNase V1 and S1 Nuclease probed samples (Additional file : Table S1).The raw single-end reads generated by Ion Proton sequencing were trimmed with BWA algorithm at a threshold of Q13 (p-value = 0.05) and length-sorted with a threshold of 25 nucleotides as implemented by SolexaQA version 2.2 [ ]. .. The pre-processed reads were mapped back to the zebrafish transcriptome assembly downloaded from Ensembl (v79, Zv9) comprising of 56,754 transcripts (33,737 genes) using a two-stepped approach involving STAR aligner [ ] and Bowtie2 [ ] as prescribed by Life technologies ( http://www.thermofisher.com/order/catalog/product/4476610?ICID=search-product & CID=fl-ion-proton-docs ).

    Expressing:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: For experiments with cells expressing photolyases, upon UV irradiation, the cells were incubated with 200 μM IdU in PBS+ supplemented with 5% FBS for 60 min at RT on the photoreactivation apparatus. .. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Modification:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: Paragraph title: ssDNA detection by modified neutral comet assay ... The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Hybridization:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: The mixture was overlaid with 5 μl of paraffin oil, denatured at 100°C for 3 min, and then incubated at the melting temperature ( Tm ) calculated for each probe on the basis of the following formula: Tm = 81.5 + 0.5 (%GC) + 16.6 (the natural log of the Na concentration) − 0.6 (% formamide). .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. Samples were then extracted once with phenol-chloroform, ethanol precipitated, resuspended in 5 μl of sequencing loading buffer , and subjected to 6% urea polyacrylamide gel electrophoresis.

    High Performance Liquid Chromatography:

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    Gas Chromatography:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Southern Blot:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. First dimension electrophoresis in 0.28% agarose was run without ethidium bromide at 1.7 V/cm at room temperature for 24 h. After the first dimension the gel was stained with ethidium bromide (300 ng/ml in TBE) and documented.

    Northern Blot:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: Paragraph title: Primer extension, S1 nuclease mapping and Northern blotting. ... After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Generated:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: FFPE libraries were generated from 100 to 200 ng DNA/total nucleic acid (TNA) using eight cycles of PCR. .. S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Further, the poly-A pool was folded in RNA Structure Buffer (Life Technologies, USA) containing 10 mM Tris pH 7, 100 mM KCl, and 10 mM MgCl2 slowly from 4 °C to 28 °C for 25 min. Each 2 μg of Poly-A RNA was digested with 10 μl (0.000125 U) of RNase V1 (Life Technologies, USA) for 45 s and 10 μl (10,000 U) of S1 Nuclease (Thermo Fisher Scientific, USA) for 10 min to achieve single hit kinetics. .. Enzyme-cleaved fragments were further purified using equal volume (100 μl) of phenol: chloroform: isoamyl alcohol (Invitrogen, USA) at 13,000 rpm (4 °C) for 10 min. RNase V1 cleaved RNA pool in the top aqueous layer was extracted and 20 μl of Inactivation/Precipitation buffer (Life Technologies, USA) was added, followed by 1 h (h) incubation at -80 °C.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: The libraries were sequenced on Ion Proton platform (Life Technologies, CA, US) employing semiconductor based chemistry after quality check to generate single end reads. .. Sequencing was done for five technical replicates each for RNase V1 and S1 Nuclease probed samples (Additional file : Table S1).The raw single-end reads generated by Ion Proton sequencing were trimmed with BWA algorithm at a threshold of Q13 (p-value = 0.05) and length-sorted with a threshold of 25 nucleotides as implemented by SolexaQA version 2.2 [ ]. .. The pre-processed reads were mapped back to the zebrafish transcriptome assembly downloaded from Ensembl (v79, Zv9) comprising of 56,754 transcripts (33,737 genes) using a two-stepped approach involving STAR aligner [ ] and Bowtie2 [ ] as prescribed by Life technologies ( http://www.thermofisher.com/order/catalog/product/4476610?ICID=search-product & CID=fl-ion-proton-docs ).

    other:

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: The only difference is that in the previous study, the embedded bacterial cells were digested with the selected restriction enzyme, whereas in this study, the cells were digested with S1 nuclease.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Additional file 1: Supplementary Tables and Figures. (PDF 454 kb) Additional file 2: Load score and percentage coverage of 54,083 transcripts. (TXT 3635 kb) Additional file 3: Multi-conformation position counts in transcripts with overlapping peaks. (TXT 712 kb) Additional file 4: Number of read starts for every position covered in RNase V1 sample. (TXT 32051 kb) Additional file 5: Number of read starts for every position covered in S1 nuclease sample. (TXT 45980 kb) Additional file 6: Total number of read starts for every position in both RNase V1 and S1 nuclease sample. (TXT 64960 kb) Additional file 7: Positions with ratio score more than one in V1 dataset. (TXT 15410 kb) Additional file 8: Positions with ratio score more than one in S1 dataset. (TXT 15865 kb) Additional file 9: PARS scores of 54,083 transcripts and non-coding RNAs. (TXT 35418 kb)

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Ratio score for every position in each of the RNase V1 (henceforth represented as V1 dataset) and S1 Nuclease (henceforth represented as S1 dataset) datasets were calculated by read start coverage for each nucleotide relative to the load of the transcript.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Folded RNA was subjected to digestion with RNase V1 (1:1600 U for 45 s) and S1 Nuclease (1:100 U for 1 min) at 28 °C respectively.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: HOTAIR was folded and probed at 37 °C with 10 μl of (1:1600 U) of RNase V1 for 45 s and 10 μl of (10,000 U) S1 Nuclease for 5 min.

    Imaging:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Polymerase Chain Reaction:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: FFPE libraries were generated from 100 to 200 ng DNA/total nucleic acid (TNA) using eight cycles of PCR. .. S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: The sodB probe, a 292-bp fragment, was amplified by PCR with Sod1 and Sod2 primers (Table ), end labeled, digested with EcoRI, and purified by using Chromaspin TE-100 columns. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Pulsed-Field Gel:

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: Paragraph title: S1 nuclease and restriction enzyme digestion of DNA in plugs and pulsed-field gel electrophoresis (PFGE) ... For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in .

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: S1 nuclease treatment can convert the supercoiled plasmids into full-length linear molecules. .. When the bacteria harboring plasmid embedded in agarose are digested with S1 nuclease followed by pulsed-field gel electrophoresis (PFGE), plasmids can be detected and their sizes can be estimated with appropriate linear DNA markers [ ]. .. In this study, 15 Chinese epidemic Leptospira strains were subjected to S1-PFGE, a distinct approach to detect the presence and the size of plasmids within Leptospira cells.

    Nucleic Acid Electrophoresis:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: Lesions were resolved in a single gel electrophoresis assay (comet assay) as previously described ( ) with modifications for the use of S1 nuclease. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Fluorescence:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Irradiation:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: For experiments with cells expressing photolyases, upon UV irradiation, the cells were incubated with 200 μM IdU in PBS+ supplemented with 5% FBS for 60 min at RT on the photoreactivation apparatus. .. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC.

    Isolation:

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Paragraph title: RNA isolation ... RNA pool of Poly-A transcripts was divided into two parts (2 μg each) for individual catalysis by RNase V1 (Life Technologies, USA) and S1 nuclease (Thermo Fisher Scientific, USA).

    Microscopy:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC. .. For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC.

    Purification:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions. .. Following treatment, DNA was purified using magnetic beads (PCR Clean-DX magnetic beads from Aline Biosciences; Catalog# C-1003–450) at a ratio of 1.8 (beads):1 (reaction).

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Further, the poly-A pool was folded in RNA Structure Buffer (Life Technologies, USA) containing 10 mM Tris pH 7, 100 mM KCl, and 10 mM MgCl2 slowly from 4 °C to 28 °C for 25 min. Each 2 μg of Poly-A RNA was digested with 10 μl (0.000125 U) of RNase V1 (Life Technologies, USA) for 45 s and 10 μl (10,000 U) of S1 Nuclease (Thermo Fisher Scientific, USA) for 10 min to achieve single hit kinetics. .. Enzyme-cleaved fragments were further purified using equal volume (100 μl) of phenol: chloroform: isoamyl alcohol (Invitrogen, USA) at 13,000 rpm (4 °C) for 10 min. RNase V1 cleaved RNA pool in the top aqueous layer was extracted and 20 μl of Inactivation/Precipitation buffer (Life Technologies, USA) was added, followed by 1 h (h) incubation at -80 °C.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: The sodB probe, a 292-bp fragment, was amplified by PCR with Sod1 and Sod2 primers (Table ), end labeled, digested with EcoRI, and purified by using Chromaspin TE-100 columns. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Sequencing:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: Paragraph title: Genomic DNA libraries and sequencing ... S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: Approximately, one picomole of the above mentioned in-vitro synthesised transcripts were pooled with zebrafish poly-A RNA to constitute 2 μg of the starting material. .. They were enzymatically probed by both RNase V1 and S1 nuclease as mentioned above and RNA libraries were prepared for sequencing. .. The data analysis was performed for the candidate ncRNAs using the pipeline described previously.

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features
    Article Snippet: The libraries were sequenced on Ion Proton platform (Life Technologies, CA, US) employing semiconductor based chemistry after quality check to generate single end reads. .. Sequencing was done for five technical replicates each for RNase V1 and S1 Nuclease probed samples (Additional file : Table S1).The raw single-end reads generated by Ion Proton sequencing were trimmed with BWA algorithm at a threshold of Q13 (p-value = 0.05) and length-sorted with a threshold of 25 nucleotides as implemented by SolexaQA version 2.2 [ ]. .. The pre-processed reads were mapped back to the zebrafish transcriptome assembly downloaded from Ensembl (v79, Zv9) comprising of 56,754 transcripts (33,737 genes) using a two-stepped approach involving STAR aligner [ ] and Bowtie2 [ ] as prescribed by Life technologies ( http://www.thermofisher.com/order/catalog/product/4476610?ICID=search-product & CID=fl-ion-proton-docs ).

    Labeling:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: Approximately 20 fmol of labeled probe was coprecipitated with either 10 or 15 μg of total RNA and then resuspended in 20 μl of hybridization buffer (80% formamide, 60 mM Tris-HCl [pH 7.5], 400 mM NaCl, 0.4 mM EDTA). .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase
    Article Snippet: The purity, integrity, and concentration of RNA template stocks were confirmed by denaturing PAGE with SYBR Gold staining. .. S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer.

    Staining:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. The control (S1 untreated) sample was subject to the same incubation conditions with identical buffers, in order to rule out possible changes in RIs due purely to the conditions.

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in . .. Electrophoresis with linear ramp time from 5 to 65 s at a gradient of 6 V/cm and an included angle of 120° was performed for 20 h to separate the DNA fragments, and gels were cooled continuously at 14°C during the running process.

    Article Title: Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase
    Article Snippet: The purity, integrity, and concentration of RNA template stocks were confirmed by denaturing PAGE with SYBR Gold staining. .. S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer.

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Neutral Comet Assay:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: Paragraph title: ssDNA detection by modified neutral comet assay ... The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Plasmid Preparation:

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: S1 nuclease treatment can convert the supercoiled plasmids into full-length linear molecules. .. When the bacteria harboring plasmid embedded in agarose are digested with S1 nuclease followed by pulsed-field gel electrophoresis (PFGE), plasmids can be detected and their sizes can be estimated with appropriate linear DNA markers [ ]. .. In this study, 15 Chinese epidemic Leptospira strains were subjected to S1-PFGE, a distinct approach to detect the presence and the size of plasmids within Leptospira cells.

    Software:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: For experiments with the ssDNA-specific S1 endonuclease, after an IdU pulse, the cells were treated with CSK100 buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 , 10 mM MOPS, 0.5% Triton X-100) for 10 min at RT, then incubated with S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate pH 4.6, 10 mM zinc acetate and 5% glycerol) with or without S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml for 30 min at 37ºC. .. DNA fibers were imaged using a fluorescent microscope (Axiovert 200, Zeiss, Jena, Germany) at a magnification of 1000×.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. Samples were then extracted once with phenol-chloroform, ethanol precipitated, resuspended in 5 μl of sequencing loading buffer , and subjected to 6% urea polyacrylamide gel electrophoresis.

    DNA Hybridization:

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    SYBR Green Assay:

    Article Title: Label-Free and Sensitive Fluorescent Detection of Sequence-Specific Single-Strand DNA Based on S1 Nuclease Cleavage Effects
    Article Snippet: The fluorescent dyes SYBR Green II (10,000× concentrated), SYBR Gold (10,000× concentrated), and PicoGreen dsDNA Reagent and Kits (200× concentrated, including 100 µg·mL−1 Lambda DNA standard) were purchased from Invitrogen (CA, USA). .. S1 nuclease was obtained from Fermentas (Beijing, China) and Takara Biotechnology Co., Ltd (Dalian, China).

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA
    Article Snippet: After centrifugation at 16,000 × g max for 30 min at 4 °C, the DNA pellet was dissolved overnight in TE buffer (10 mm Tris/HCl and 1 mm EDTA, pH 7.8) at 55 °C. mtDNA was quantified by qPCR using primers against ND5 with RpL32 as an internal standard (see Table S1 ) in a StepOnePlus Real-Time PCR System with a Fast SYBR Green Master Mix kit. .. For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min.

    Selection:

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples
    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions. .. Following treatment, DNA was purified using magnetic beads (PCR Clean-DX magnetic beads from Aline Biosciences; Catalog# C-1003–450) at a ratio of 1.8 (beads):1 (reaction).

    Agarose Gel Electrophoresis:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. First dimension electrophoresis in 0.28% agarose was run without ethidium bromide at 1.7 V/cm at room temperature for 24 h. After the first dimension the gel was stained with ethidium bromide (300 ng/ml in TBE) and documented.

    Electrophoresis:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. The control (S1 untreated) sample was subject to the same incubation conditions with identical buffers, in order to rule out possible changes in RIs due purely to the conditions.

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species
    Article Snippet: For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in . .. For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in .

    Ethanol Precipitation:

    Article Title: Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism
    Article Snippet: Restriction endonuclease (Fermentas) reactions were incubated at 37°C for 4 h with 4 units of enzyme per µg of mitochondrial nucleic acid, in manufacturer’s recommended buffers. .. Any subsequent treatment with S1 nuclease (Fermentas) was also carried out according to the enzyme manufacturer’s protocol, after stopping the initial restriction digest and removing enzyme by phenol/chloroform extraction and ethanol precipitation. .. S1 nuclease digestion (0.2 u per µg of nucleic acid) was for 2 min at room temperature.

    Concentration Assay:

    Article Title: Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase
    Article Snippet: S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer. .. S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis
    Article Snippet: After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C. .. After 4 to 16 h of hybridization, 180 μl of ice-cold S1 buffer (33 mM sodium acetate [pH 5.2], 5 mM ZnSO4 , 250 mM NaCl) and 100 U of S1 nuclease (Invitrogen) were added, and S1 digestion was carried out for 30 min at 37°C.

    Migration:

    Article Title: Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase
    Article Snippet: S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer. .. S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer.

    Single Cell Gel Electrophoresis:

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells
    Article Snippet: Lesions were resolved in a single gel electrophoresis assay (comet assay) as previously described ( ) with modifications for the use of S1 nuclease. .. The comet slides were washed in S1 nuclease buffer (50 mM NaCl, 30 mM sodium acetate, pH 4.6 and 5% glycerol) before the addition of S1 nuclease (Invitrogen, Life Technologies) at 20 U/ml in 1× S1 nuclease buffer and 50 mM NaCl (supplied by the manufacturer) to half the slide for 30 min at 37°C.

    Activity Assay:

    Article Title: Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase
    Article Snippet: Paragraph title: Template Preparation and Activity Assays ... S1 nuclease (Fermentas) treatment was performed for 10–20 min at 37 °C in the manufacturer's buffer.

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    Thermo Fisher s1 nuclease
    Effects of <t>S1</t> nuclease treatment on genome coverage and sequence bias. ( A ) Genome coverage. A screen shot of an IGV image of a representative chromosomal region is shown for libraries that were prepared from fresh normal and tumor samples, and matching FormaPure FFPE samples with (F+S1) or without (F-S1) S1 nuclease treatment. The lower panel is an enlarged portion of the region shown in the upper panel. Colored vertical lines within coverage histograms designate consensus SNVs that are also shown as colored vertical lines within reads. Colored arrow boxes within reads represent SSARs and improperly paired artifacts (Red = insert size too large relative to consensus insert size range; Blue = insert too small; Green = SSARs. Other colors depict paired reads that aligned to regions from different chromosomes). ( B ) Effects of S1 nuclease treatment on GC-bias. Samples are the same as in (A). Upper panel shows normalized coverage data at various levels of GC-content and lower panel shows read distribution as a function of GC-content.
    S1 Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of S1 nuclease treatment on genome coverage and sequence bias. ( A ) Genome coverage. A screen shot of an IGV image of a representative chromosomal region is shown for libraries that were prepared from fresh normal and tumor samples, and matching FormaPure FFPE samples with (F+S1) or without (F-S1) S1 nuclease treatment. The lower panel is an enlarged portion of the region shown in the upper panel. Colored vertical lines within coverage histograms designate consensus SNVs that are also shown as colored vertical lines within reads. Colored arrow boxes within reads represent SSARs and improperly paired artifacts (Red = insert size too large relative to consensus insert size range; Blue = insert too small; Green = SSARs. Other colors depict paired reads that aligned to regions from different chromosomes). ( B ) Effects of S1 nuclease treatment on GC-bias. Samples are the same as in (A). Upper panel shows normalized coverage data at various levels of GC-content and lower panel shows read distribution as a function of GC-content.

    Journal: Nucleic Acids Research

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples

    doi: 10.1093/nar/gky1142

    Figure Lengend Snippet: Effects of S1 nuclease treatment on genome coverage and sequence bias. ( A ) Genome coverage. A screen shot of an IGV image of a representative chromosomal region is shown for libraries that were prepared from fresh normal and tumor samples, and matching FormaPure FFPE samples with (F+S1) or without (F-S1) S1 nuclease treatment. The lower panel is an enlarged portion of the region shown in the upper panel. Colored vertical lines within coverage histograms designate consensus SNVs that are also shown as colored vertical lines within reads. Colored arrow boxes within reads represent SSARs and improperly paired artifacts (Red = insert size too large relative to consensus insert size range; Blue = insert too small; Green = SSARs. Other colors depict paired reads that aligned to regions from different chromosomes). ( B ) Effects of S1 nuclease treatment on GC-bias. Samples are the same as in (A). Upper panel shows normalized coverage data at various levels of GC-content and lower panel shows read distribution as a function of GC-content.

    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Techniques: Sequencing, Formalin-fixed Paraffin-Embedded, Gas Chromatography

    Effects of S1 nuclease treatment on FFPE-associated somatic SNV noise. Libraries were prepared from fresh normal and tumor samples from the same patient, and matching FormaPure FFPE samples with (F+S1) and or without (F-S1) S1 nuclease treatment. For each of the three latter libraries, SNVs were identified relative to the library from the normal blood sample. Upset plots indicating data overlaps are shown. In ( A ) are data obtained using a QSS score cutoff ≥15 as a cut-off. In ( B ) are data generated using a QSS score cutoff ≥35.

    Journal: Nucleic Acids Research

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples

    doi: 10.1093/nar/gky1142

    Figure Lengend Snippet: Effects of S1 nuclease treatment on FFPE-associated somatic SNV noise. Libraries were prepared from fresh normal and tumor samples from the same patient, and matching FormaPure FFPE samples with (F+S1) and or without (F-S1) S1 nuclease treatment. For each of the three latter libraries, SNVs were identified relative to the library from the normal blood sample. Upset plots indicating data overlaps are shown. In ( A ) are data obtained using a QSS score cutoff ≥15 as a cut-off. In ( B ) are data generated using a QSS score cutoff ≥35.

    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Techniques: Formalin-fixed Paraffin-Embedded, Generated

    SSAR mapping and diagrammatic depiction of the proposed mechanism. The SSAR example shown is a screen shot of an actual IGV image. At the top (I) we depict a ds-DNA region of intact gDNA. In the process of FFPE preparation, storage and extraction (II), gDNA is fragmented and denatured. In the absence of S1 nuclease (III left), ss-DNA fragments from non-contiguous regions of the genome anneal via short complementary repetitive sequences (red asterisks). In contrast, ss-DNA fragments and overhangs are removed upon treatment with S1 nuclease (III right). During the end-repair step of library construction, T4 DNA polymerase removes overhangs (IV) and fills ends (V), resulting in the formation of double-stranded chimeric fragments (‘A’ in V). One class of such chimeric fragments yield SSARs (‘A’ in VI). R1 = read; R2 = read 2. For SSARs, part of Read 2 aligns in the expected paired-end orientation while the distal end of Read 2 does not match the reference at that position and instead aligns to a nearby region of the reference genome in the opposite orientation (denoted as R2′).

    Journal: Nucleic Acids Research

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples

    doi: 10.1093/nar/gky1142

    Figure Lengend Snippet: SSAR mapping and diagrammatic depiction of the proposed mechanism. The SSAR example shown is a screen shot of an actual IGV image. At the top (I) we depict a ds-DNA region of intact gDNA. In the process of FFPE preparation, storage and extraction (II), gDNA is fragmented and denatured. In the absence of S1 nuclease (III left), ss-DNA fragments from non-contiguous regions of the genome anneal via short complementary repetitive sequences (red asterisks). In contrast, ss-DNA fragments and overhangs are removed upon treatment with S1 nuclease (III right). During the end-repair step of library construction, T4 DNA polymerase removes overhangs (IV) and fills ends (V), resulting in the formation of double-stranded chimeric fragments (‘A’ in V). One class of such chimeric fragments yield SSARs (‘A’ in VI). R1 = read; R2 = read 2. For SSARs, part of Read 2 aligns in the expected paired-end orientation while the distal end of Read 2 does not match the reference at that position and instead aligns to a nearby region of the reference genome in the opposite orientation (denoted as R2′).

    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Techniques: Formalin-fixed Paraffin-Embedded

    Effects of S1 nuclease treatment on FFPE-associated CNV noise. ( A ) Example illustrating CNV noise. Samples are the same as in Figure 5 . Using a bin size of 200 reads, CNV segments were calculated in the tumor samples relative to the normal blood sample and the resulting profile is shown for chromosome 14. ( B ) CNV counts at the gene level. Gains are shown on the left and losses are shown in the middle panel. Jaccard's intersection index (Materials and Methods) is shown in the right panel as a measure of overlap of gene-level CNVs between the three samples.

    Journal: Nucleic Acids Research

    Article Title: Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples

    doi: 10.1093/nar/gky1142

    Figure Lengend Snippet: Effects of S1 nuclease treatment on FFPE-associated CNV noise. ( A ) Example illustrating CNV noise. Samples are the same as in Figure 5 . Using a bin size of 200 reads, CNV segments were calculated in the tumor samples relative to the normal blood sample and the resulting profile is shown for chromosome 14. ( B ) CNV counts at the gene level. Gains are shown on the left and losses are shown in the middle panel. Jaccard's intersection index (Materials and Methods) is shown in the right panel as a measure of overlap of gene-level CNVs between the three samples.

    Article Snippet: S1 Nuclease was purchased from Thermo Fisher Scientific (Catalog# MAN0013722) and the treatment of 100–300 ng gDNA or TNA was as described in the manufacturer's instructions.

    Techniques: Formalin-fixed Paraffin-Embedded

    Identification of homologous sequence of lcp1- rep , lcp2- rep  and lcp3- rep  in 15 Chinese epidemic  Leptospira  strains. Leptospira  strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions, blotted to a nylon membrane and then hybridized with the probes of lcp1- rep (A), lcp2- rep (B) and lcp3- rep (C). Probes were generated by PCR of T-vector plasmid DNA containing  rep  genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively (  S2 Table ). Markers are shown on the right. 56601–56655 represent 15  Leptospira  Chinese epidemic strains as detailed in   Table 3 .

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    doi: 10.1371/journal.pntd.0004220

    Figure Lengend Snippet: Identification of homologous sequence of lcp1- rep , lcp2- rep and lcp3- rep in 15 Chinese epidemic Leptospira strains. Leptospira strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions, blotted to a nylon membrane and then hybridized with the probes of lcp1- rep (A), lcp2- rep (B) and lcp3- rep (C). Probes were generated by PCR of T-vector plasmid DNA containing rep genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively ( S2 Table ). Markers are shown on the right. 56601–56655 represent 15 Leptospira Chinese epidemic strains as detailed in Table 3 .

    Article Snippet: For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in .

    Techniques: Sequencing, Generated, Polymerase Chain Reaction, Plasmid Preparation

    Detection of lbp1 and lbp2 plasmids by PFGE. ( A) S1-PFGE-based Southern blot analysis. (B) Single restriction enzyme digestion of the plasmids followed by Southern blot analysis. M represents the standard strain  Salmonella enterica  serotype Braenderup (H9812) digested with  Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. ND, undigested  L .  borgpetersenii  serovar Ballum strain 56604;  L .  borgpetersenii  serovar Ballum strain 56604 were digested by S1 nuclease,  Not I and  Pst I. For Southern blot analysis, the genomic DNA of  L .  borgpetersenii  serovar Ballum strain 56604 was blotted to a nylon membrane and hybridized by lcp1- rep  and lcp2- rep . Probes were generated by PCR of T-vector plasmid DNA containing  rep  genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively (  S2 Table ).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    doi: 10.1371/journal.pntd.0004220

    Figure Lengend Snippet: Detection of lbp1 and lbp2 plasmids by PFGE. ( A) S1-PFGE-based Southern blot analysis. (B) Single restriction enzyme digestion of the plasmids followed by Southern blot analysis. M represents the standard strain Salmonella enterica serotype Braenderup (H9812) digested with Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. ND, undigested L . borgpetersenii serovar Ballum strain 56604; L . borgpetersenii serovar Ballum strain 56604 were digested by S1 nuclease, Not I and Pst I. For Southern blot analysis, the genomic DNA of L . borgpetersenii serovar Ballum strain 56604 was blotted to a nylon membrane and hybridized by lcp1- rep and lcp2- rep . Probes were generated by PCR of T-vector plasmid DNA containing rep genes with primer pairs of lcp1- rep -probe FR, lcp2- rep -probe-FR and lcp3- rep -probe-FR, respectively ( S2 Table ).

    Article Snippet: For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in .

    Techniques: Southern Blot, Molecular Weight, Marker, Generated, Polymerase Chain Reaction, Plasmid Preparation

    Plasmids detection in 15 Chinese epidemic  Leptospira  strains by S1-PFGE separation. Leptospira  strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions. Marker represents the pattern of the standard strain  Salmonella enterica  serotype Braenderup (H9812) digested with  Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. M represents the marker electrophoresed with each sample. 56601–56655 represent 15 Chinese epidemic  Leptospira  strains as detailed in   Table 3 .

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Distribution of Plasmids in Distinct Leptospira Pathogenic Species

    doi: 10.1371/journal.pntd.0004220

    Figure Lengend Snippet: Plasmids detection in 15 Chinese epidemic Leptospira strains by S1-PFGE separation. Leptospira strains were embedded in agarose, lysed and digested with S1 nuclease and electrophoresed under pulsed-field conditions. Marker represents the pattern of the standard strain Salmonella enterica serotype Braenderup (H9812) digested with Xba I electrophoresed under pulsed-field conditions as a molecular weight marker. M represents the marker electrophoresed with each sample. 56601–56655 represent 15 Chinese epidemic Leptospira strains as detailed in Table 3 .

    Article Snippet: For different Leptospira species, the digestion reaction was similar in time but different in the enzyme added (S1 nuclease, Thermo Scientific; Not I and Pst I, New England Biolabs), as shown in .

    Techniques: Marker, Molecular Weight