exonuclease i  (New England Biolabs)


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    Name:
    Exonuclease I (E.coli) - 1
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    Catalog Number:
    M0293L
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    85
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    Structured Review

    New England Biolabs exonuclease i
    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

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    Images

    1) Product Images from "Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals"

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091457

    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.
    Figure Legend Snippet: Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Techniques Used: Hybridization, Nucleic Acid Electrophoresis, Electrophoresis, Blocking Assay, Incubation, Labeling

    Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk  with 1 pmol  miD156s  at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk  with 1 pmol  miD156s  at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk  with 1 pmol  miD156s  at different temperatures by traditional Northern hybridization.  miD156  marked “+”,  miD156  with one-base mismatch marked “−1”,  miD156  with three-base mismatch marked “−3” and  miD156  with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of  miD156 ; 10f represents 10 fmol of  miD156 ; 5f represents 5 fmol of  miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .
    Figure Legend Snippet: Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Techniques Used: Northern Blot, Hybridization

    2) Product Images from "The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein"

    Article Title: The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein

    Journal:

    doi: 10.1128/IAI.00365-13

    Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane
    Figure Legend Snippet: Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane

    Techniques Used: Agarose Gel Electrophoresis

    3) Product Images from "A convenient system for highly specific and sensitive detection of miRNA expression"

    Article Title: A convenient system for highly specific and sensitive detection of miRNA expression

    Journal:

    doi: 10.1261/rna.040220.113

    Schematic diagram of procedures. Extracted RNAs are hybridized in buffer with special hairpin DNA probes. Next, FD BamH I is added into the mixture to cut the probes. Following that, exonuclease I is pipetted into the mixture to digest the nonhybridized
    Figure Legend Snippet: Schematic diagram of procedures. Extracted RNAs are hybridized in buffer with special hairpin DNA probes. Next, FD BamH I is added into the mixture to cut the probes. Following that, exonuclease I is pipetted into the mixture to digest the nonhybridized

    Techniques Used:

    4) Product Images from "Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA"

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016925

    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase.  B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All.  C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    Figure Legend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    5) Product Images from "The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein"

    Article Title: The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein

    Journal:

    doi: 10.1128/IAI.00365-13

    Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane
    Figure Legend Snippet: Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane

    Techniques Used: Agarose Gel Electrophoresis

    6) Product Images from "Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex"

    Article Title: Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks305

    Functional significance of the RSfp motif in Sfr1. ( A ) Sfr1 (303 residues) harbors a RSfp motif in its N-terminus. This motif is deleted in the dN104Sfr1 mutant. ( B ) Purified Swi5–dN104Sfr1 complex (1.5 µg) was subjected to 13% SDS–PAGE and Coomassie Blue staining. ( C ) Purified Swi5–dN104Sfr1 mutant complex was tested for Rad51 interaction by affinity pulldown through the (His) 6  tag on dN104Sfr1. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. ( D ) DNA strand exchange was conducted with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. ( E ) The stability of the Rad51 presynaptic filament was tested by the exonuclease I assay with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, Swi5–Sfr1; S5-dN104S1, Swi5–dN104Sfr1.
    Figure Legend Snippet: Functional significance of the RSfp motif in Sfr1. ( A ) Sfr1 (303 residues) harbors a RSfp motif in its N-terminus. This motif is deleted in the dN104Sfr1 mutant. ( B ) Purified Swi5–dN104Sfr1 complex (1.5 µg) was subjected to 13% SDS–PAGE and Coomassie Blue staining. ( C ) Purified Swi5–dN104Sfr1 mutant complex was tested for Rad51 interaction by affinity pulldown through the (His) 6 tag on dN104Sfr1. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. ( D ) DNA strand exchange was conducted with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. ( E ) The stability of the Rad51 presynaptic filament was tested by the exonuclease I assay with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, Swi5–Sfr1; S5-dN104S1, Swi5–dN104Sfr1.

    Techniques Used: Functional Assay, Mutagenesis, Purification, SDS Page, Staining, Concentration Assay, Standard Deviation

    Stabilization of the Rad51 presynaptic filament by Swi5–Sfr1. ( A ) Schematic of the exonuclease I protection assay for examining presynaptic filament stability. The Rad51 presynaptic filament harboring 5′- 32 P-labeled DNA is incubated with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The  32 P label is denoted by the asterisk. ( B ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5–Sfr1. The results were graphed. ( C ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5 (panel I), Sfr1 (panel II) or Swi5–Sfr1. The results were graphed. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.
    Figure Legend Snippet: Stabilization of the Rad51 presynaptic filament by Swi5–Sfr1. ( A ) Schematic of the exonuclease I protection assay for examining presynaptic filament stability. The Rad51 presynaptic filament harboring 5′- 32 P-labeled DNA is incubated with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( B ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5–Sfr1. The results were graphed. ( C ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5 (panel I), Sfr1 (panel II) or Swi5–Sfr1. The results were graphed. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Standard Deviation

    7) Product Images from "Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing"

    Article Title: Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1229

    PSF stimulates dsDNA capture by the DMC1–ssDNA complex. ( A ) Schematic representation of the dsDNA capturing assay. Asterisks indicate the  32 P-labeled ends of the dsDNA. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), and  32 P-labeled dsDNA 49-mer (1.5 µM) in the presence of the 83-mer poly dT ssDNA conjugated to the beads. The dsDNA captured by the DMC1–ssDNA complex was detected. Lane 1 indicates a negative control reaction without the proteins. Lanes 2–4 represent the experiments with DMC1 alone, PSF alone, and both DMC1 and PSF, respectively. Portions including 80% of the dsDNA recovered in the ssDNA bound fraction were analyzed by native PAGE (upper panel), and 10% portions of the dsDNA samples remaining in the ssDNA unbound fraction were analyzed by native PAGE (lower panel). ( C ) The ssDNA protection assay with DMC1. The DMC1–ssDNA complexes were treated with exonuclease I in the presence or absence of PSF. The resulting ssDNAs were analyzed by native PAGE. Lane 1 indicates a negative control experiment without proteins and exonuclease I. Lanes 3, 6, 9 and 12 indicate experiments with 0.3 µM PSF, and lanes 4, 7, 10 and 13 indicate experiments with 0.6 µM PSF. Lanes 2, 5, 8 and 11 indicate experiments without PSF. The DMC1 concentrations were 0 µM (lanes 2–4), 0.5 µM (lanes 5–7), 1 µM (lanes 8–10) and 2 µM (lanes 11–13). ( D ) The ssDNA protection assay with RAD51. The reaction was conducted by the same method as in panel C, except RAD51 was used instead of DMC1. ( E ) Schematic representation of the synaptic complex formation assay. ( F ) The synaptic complex formation reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), in the presence of the 70-mer ssDNA and superhelical dsDNA. Lane 1 indicates a negative control experiment without proteins and PstI restriction enzyme. Lanes 2 and 6 represent the experiments without proteins. Lanes 2–5 and 6–9 indicate experiments with the homologous ssDNA and heterologous ssDNA, respectively. Lanes 3 and 7 indicate the experiments with DMC1 alone. Lanes 4 and 8 indicate the experiments with PSF alone. Lanes 5 and 9 indicate the experiments with both DMC1 and PSF. NC and SC represent nicked circular and superhelical dsDNA molecules. ( G ) Graphic representation of the synaptic complex formation assay shown in panel F. The band intensities of the protected DNAs were quantitated, and the average values of three independent experiments are shown with the SD values.
    Figure Legend Snippet: PSF stimulates dsDNA capture by the DMC1–ssDNA complex. ( A ) Schematic representation of the dsDNA capturing assay. Asterisks indicate the 32 P-labeled ends of the dsDNA. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), and 32 P-labeled dsDNA 49-mer (1.5 µM) in the presence of the 83-mer poly dT ssDNA conjugated to the beads. The dsDNA captured by the DMC1–ssDNA complex was detected. Lane 1 indicates a negative control reaction without the proteins. Lanes 2–4 represent the experiments with DMC1 alone, PSF alone, and both DMC1 and PSF, respectively. Portions including 80% of the dsDNA recovered in the ssDNA bound fraction were analyzed by native PAGE (upper panel), and 10% portions of the dsDNA samples remaining in the ssDNA unbound fraction were analyzed by native PAGE (lower panel). ( C ) The ssDNA protection assay with DMC1. The DMC1–ssDNA complexes were treated with exonuclease I in the presence or absence of PSF. The resulting ssDNAs were analyzed by native PAGE. Lane 1 indicates a negative control experiment without proteins and exonuclease I. Lanes 3, 6, 9 and 12 indicate experiments with 0.3 µM PSF, and lanes 4, 7, 10 and 13 indicate experiments with 0.6 µM PSF. Lanes 2, 5, 8 and 11 indicate experiments without PSF. The DMC1 concentrations were 0 µM (lanes 2–4), 0.5 µM (lanes 5–7), 1 µM (lanes 8–10) and 2 µM (lanes 11–13). ( D ) The ssDNA protection assay with RAD51. The reaction was conducted by the same method as in panel C, except RAD51 was used instead of DMC1. ( E ) Schematic representation of the synaptic complex formation assay. ( F ) The synaptic complex formation reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), in the presence of the 70-mer ssDNA and superhelical dsDNA. Lane 1 indicates a negative control experiment without proteins and PstI restriction enzyme. Lanes 2 and 6 represent the experiments without proteins. Lanes 2–5 and 6–9 indicate experiments with the homologous ssDNA and heterologous ssDNA, respectively. Lanes 3 and 7 indicate the experiments with DMC1 alone. Lanes 4 and 8 indicate the experiments with PSF alone. Lanes 5 and 9 indicate the experiments with both DMC1 and PSF. NC and SC represent nicked circular and superhelical dsDNA molecules. ( G ) Graphic representation of the synaptic complex formation assay shown in panel F. The band intensities of the protected DNAs were quantitated, and the average values of three independent experiments are shown with the SD values.

    Techniques Used: Capturing Assay, Labeling, Negative Control, Clear Native PAGE, Tube Formation Assay

    8) Product Images from "Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System"

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014388

    Use of Species-Independent Translational Sequence (SITS) in Overlap Extension (OE) PCR-mediated assembly of templates for  in vitro  translation. (A) Schematic representation of SITS structure. (B) Scheme of purification-free OE-PCR for synthesis of DNA templates for cell-free translation. PCR amplification of individual fragments with partially overlapping sequences. 5′ NTR – 5′ not transcribed regions, 3′ UTR – 3′ untranslated region. (C) Removal of residual primers from PCR reaction by exonuclease I treatment. (D) Fragments are fused and amplified by Overlap Extension PCR in the presence of the flanking primers.
    Figure Legend Snippet: Use of Species-Independent Translational Sequence (SITS) in Overlap Extension (OE) PCR-mediated assembly of templates for in vitro translation. (A) Schematic representation of SITS structure. (B) Scheme of purification-free OE-PCR for synthesis of DNA templates for cell-free translation. PCR amplification of individual fragments with partially overlapping sequences. 5′ NTR – 5′ not transcribed regions, 3′ UTR – 3′ untranslated region. (C) Removal of residual primers from PCR reaction by exonuclease I treatment. (D) Fragments are fused and amplified by Overlap Extension PCR in the presence of the flanking primers.

    Techniques Used: Sequencing, Overlap Extension Polymerase Chain Reaction, In Vitro, Purification, Polymerase Chain Reaction, Amplification

    9) Product Images from "MLGA--a rapid and cost-efficient assay for gene copy-number analysis"

    Article Title: MLGA--a rapid and cost-efficient assay for gene copy-number analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm651

    ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme.  (I)  Genomic DNA is digested by restriction enzyme to generate targets with defined ends.  (II)  Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization.  (III)  To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I).  (IV)  Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.
    Figure Legend Snippet: ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.

    Techniques Used: Multiplex Assay, Ligation, Amplification, Plasmid Preparation, Hybridization, Polymerase Chain Reaction, Sequencing, Electrophoresis

    10) Product Images from "Role of the RAD51–SWI5–SFR1 Ensemble in homologous recombination"

    Article Title: Role of the RAD51–SWI5–SFR1 Ensemble in homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw375

    Functional characterization of SWI5–SFR1 dN202  complex. ( A ) To map the minimal complex unit of SWI5–SFR1, purified SWI5–SFR1 was incubated with proteinase K for the indicated times. The proteolytic products were resolved by 15% SDS-PAGE and stained by Coomassie Blue staining. The asterisk denotes the SFR1 dN202 . ( B ) Purified SWI5–SFR1 dN202  complex (3 μg) was subjected to 15% SDS-PAGE and Coomassie Blue staining. ( C ) SWI5–SFR1 dN202  was tested for RAD51 interaction by affinity pulldown as described in Figure   1D . ( D ) DNA strand exchange assay to monitor the stimulatory effect of SWI5–SFR1 by RAD51. ( I ) Schematic of the DNA strand exchange assay. The radiolabeled substrate and product are visualized and quantified by phosphorimaging analysis after PAGE. The asterisk denotes the  32 P label. ( II ) DNA strand exchange was conducted with the indicated amounts of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. ( E ) Exonuclease I protection assay to monitor the influence of SWI5–SFR1 on the stability of RAD51 filament. ( I ) Schematic of the exonuclease I protection assay. The RAD51 filament harboring 5′- 32 P-labeled DNA is challenged with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The  32 P label is denoted by the asterisk. ( II ) Treatment of the RAD51 presynaptic filament with exonuclease I in the presence of the indicated concentrations of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. (D-E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.
    Figure Legend Snippet: Functional characterization of SWI5–SFR1 dN202 complex. ( A ) To map the minimal complex unit of SWI5–SFR1, purified SWI5–SFR1 was incubated with proteinase K for the indicated times. The proteolytic products were resolved by 15% SDS-PAGE and stained by Coomassie Blue staining. The asterisk denotes the SFR1 dN202 . ( B ) Purified SWI5–SFR1 dN202 complex (3 μg) was subjected to 15% SDS-PAGE and Coomassie Blue staining. ( C ) SWI5–SFR1 dN202 was tested for RAD51 interaction by affinity pulldown as described in Figure 1D . ( D ) DNA strand exchange assay to monitor the stimulatory effect of SWI5–SFR1 by RAD51. ( I ) Schematic of the DNA strand exchange assay. The radiolabeled substrate and product are visualized and quantified by phosphorimaging analysis after PAGE. The asterisk denotes the 32 P label. ( II ) DNA strand exchange was conducted with the indicated amounts of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. ( E ) Exonuclease I protection assay to monitor the influence of SWI5–SFR1 on the stability of RAD51 filament. ( I ) Schematic of the exonuclease I protection assay. The RAD51 filament harboring 5′- 32 P-labeled DNA is challenged with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( II ) Treatment of the RAD51 presynaptic filament with exonuclease I in the presence of the indicated concentrations of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. (D-E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

    Techniques Used: Functional Assay, Purification, Incubation, SDS Page, Staining, Polyacrylamide Gel Electrophoresis, Labeling, Standard Deviation

    SWI5 F83A/L85A-SFR1 is functionally impaired. ( A ) The effect of SWI5–SFR1 or SWI5 FL/AA –SFR1 on RAD51-mediated DNA strand exchange was examined. The results were graphed. ( B ) Exonuclease I protection assay was conducted with the indicated concentrations of SWI5–SFR1 and SWI5 FL/AA –SFR1. The results were graphed. ( C ) The average length of RAD51 filaments with negative staining was determined by electron microscopy (see Supplementary Figure S5). RAD51 was examined alone or with SWI5–SFR1 or SWI5 FL/AA –SFR1. The total 225 filaments were counted in each reaction. We note that the average length of the presynaptic filament in the presence of SWI5–SFR1 is much longer than expected. This could be due to the end-to-end association between two DNA molecules by RAD51 as described by Baumann et al. (  35 ). ( D ) Thin-layer chromatography to monitor the hydrolysis of [γ- 32 P] ATP by RAD51 in the absence or presence of indicated concentrations of SWI5–SFR1 or SWI5 FL/AA –SFR1. The results were graphed. (A, B and D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.
    Figure Legend Snippet: SWI5 F83A/L85A-SFR1 is functionally impaired. ( A ) The effect of SWI5–SFR1 or SWI5 FL/AA –SFR1 on RAD51-mediated DNA strand exchange was examined. The results were graphed. ( B ) Exonuclease I protection assay was conducted with the indicated concentrations of SWI5–SFR1 and SWI5 FL/AA –SFR1. The results were graphed. ( C ) The average length of RAD51 filaments with negative staining was determined by electron microscopy (see Supplementary Figure S5). RAD51 was examined alone or with SWI5–SFR1 or SWI5 FL/AA –SFR1. The total 225 filaments were counted in each reaction. We note that the average length of the presynaptic filament in the presence of SWI5–SFR1 is much longer than expected. This could be due to the end-to-end association between two DNA molecules by RAD51 as described by Baumann et al. ( 35 ). ( D ) Thin-layer chromatography to monitor the hydrolysis of [γ- 32 P] ATP by RAD51 in the absence or presence of indicated concentrations of SWI5–SFR1 or SWI5 FL/AA –SFR1. The results were graphed. (A, B and D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

    Techniques Used: Negative Staining, Electron Microscopy, Thin Layer Chromatography, Standard Deviation

    11) Product Images from "Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood"

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr424

    Exonuclease activity and hybridization length affects assay sensitivity.  (A)  VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo +  all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.
    Figure Legend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Techniques Used: Activity Assay, Hybridization, Generated

    12) Product Images from "Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells"

    Article Title: Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.34

    Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a ,  b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a  32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in  b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the  32 P-labeled (CCCTAA) 3  probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d  were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3).  P- values were obtained using the Student’s  t -test. ** P
    Figure Legend Snippet: Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P

    Techniques Used: Gene Knockout, Labeling, TRF Assay, Staining, DNA Extraction, Hybridization

    13) Product Images from "A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding"

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq687

    Experimental setting for genome-wide analysis of DNA-crosslinking probability mediated by TMP PB. In a typical experiment, exponentially growing yeast cells were split in three identical fractions. In the first fraction, cells were incubated with TMP and irradiated to produce a limited amount of intracellular DNA crosslinks (one crosslink per 10 kb). Cellular DNA was extracted and fragmented (2-kb average length). In the second fraction, cellular DNA was extracted, fragmented (2-kb average length) and then incubated with TMP and irradiated to produce one crosslink per 10 kb. Genomic DNA fragments from the above  in vivo  and  in vitro  experiments were thermally denatured to convert un-crosslinked segments into ssDNA. Exonuclease I degraded then ssDNA chains from their 3′-end, such that only crosslinked duplexes (dsDNA) and DNA chains with a blocked 3′-end remained. Exonuclease λ digested then ssDNA and dsDNA and from their 5′-ends. As a result, un-crosslinked fragments were degraded and crosslinked fragments were converted into a pair of ssDNA chains bridged by a TMP linkage. Random priming along these chains produced radiolabeled sequences that were hybridized on DNA arrays .  The third fraction of the yeast culture was used to conduct a genome-wide analysis of ongoing transcription (GRO).  In vivo  radiolabeled RNA was purified and hybridized on arrays of the same lot used to analyze the TMP-mediated DNA crosslinks.
    Figure Legend Snippet: Experimental setting for genome-wide analysis of DNA-crosslinking probability mediated by TMP PB. In a typical experiment, exponentially growing yeast cells were split in three identical fractions. In the first fraction, cells were incubated with TMP and irradiated to produce a limited amount of intracellular DNA crosslinks (one crosslink per 10 kb). Cellular DNA was extracted and fragmented (2-kb average length). In the second fraction, cellular DNA was extracted, fragmented (2-kb average length) and then incubated with TMP and irradiated to produce one crosslink per 10 kb. Genomic DNA fragments from the above in vivo and in vitro experiments were thermally denatured to convert un-crosslinked segments into ssDNA. Exonuclease I degraded then ssDNA chains from their 3′-end, such that only crosslinked duplexes (dsDNA) and DNA chains with a blocked 3′-end remained. Exonuclease λ digested then ssDNA and dsDNA and from their 5′-ends. As a result, un-crosslinked fragments were degraded and crosslinked fragments were converted into a pair of ssDNA chains bridged by a TMP linkage. Random priming along these chains produced radiolabeled sequences that were hybridized on DNA arrays . The third fraction of the yeast culture was used to conduct a genome-wide analysis of ongoing transcription (GRO). In vivo radiolabeled RNA was purified and hybridized on arrays of the same lot used to analyze the TMP-mediated DNA crosslinks.

    Techniques Used: Genome Wide, Incubation, Irradiation, In Vivo, In Vitro, Produced, Purification

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    Amplification:

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    Filtration:

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
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    Positive Control:

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: One three-spined stickleback individual from the Baltic Sea (60°12' N, 25°11' E) was used as a positive control. .. PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs.

    Synthesized:

    Article Title: Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity
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    TA Cloning:

    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
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    Construct:

    Article Title: Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma
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    Gene Assay:

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    Incubation:

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    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
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    Article Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
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    Article Title: Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis
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    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
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    Article Title: Mechanistic insights into the role of Hop2-Mnd1 in meiotic homologous DNA pairing
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    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction
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    Article Title: A molecular inversion probe assay for detecting alternative splicing
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    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
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    Expressing:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
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    Article Title: Natural Genetic Variation of Integrin Alpha L (Itgal) Modulates Ischemic Brain Injury in Stroke
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    Modification:

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: The genomic PCR was performed similarly to the plasmid-programmed reaction with the modification in the annealing step (temperature decrement of 1° from 68°C to 60°C on the first eight cycles was used, then 27 cycles at 60°C, annealing for 50 seconds) and prolonged to 40 seconds elongation step. .. Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes.

    Ligation:

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Following ligation, the beads were washed three times with Dynabead wash buffer: twice at 58°C and once at room temperature. .. The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S.

    Generated:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
    Article Snippet: Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min). .. Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min).

    Article Title: Natural Genetic Variation of Integrin Alpha L (Itgal) Modulates Ischemic Brain Injury in Stroke
    Article Snippet: PCR was performed using an appropriate dilution of cDNA generated from the cerebral cortex of F1 (B6×BALB) animals. .. Amplicons containing coding SNPs were amplified by conventional PCR, and 15 ul of PCR products were treated with 1 U exonuclease I (New England Biolabs) and 5 U of Shrimp Alkaline Phosphatase (SAP) (Promega).

    Cell Culture:

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    Bridge PCR:

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
    Article Snippet: This step not only makes the eluted DNA double stranded to be precisely size-selected by SPRI beads, but also synthesizes the sequence required for bridge PCR. .. We transferred the supernatant to a new tube, added 1 μl of 20 U/μl Exonuclease I (NEB) and incubated the tube at 37°C for 15 min.

    Overlap Extension Polymerase Chain Reaction:

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: Paragraph title: Preparation of PCR products for OE-PCR ... Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes.

    DNA Sequencing:

    Article Title: Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22-1 as an allele of chromatin- remodelling factor gene swr1
    Article Snippet: Paragraph title: Genomic DNA sequencing ... The resulting 2 kb PCR products were treated with exonuclease I and Antarctic phosphatase (NEB) and directly used in sequencing reactions.

    Sequencing:

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
    Article Snippet: This step not only makes the eluted DNA double stranded to be precisely size-selected by SPRI beads, but also synthesizes the sequence required for bridge PCR. .. We transferred the supernatant to a new tube, added 1 μl of 20 U/μl Exonuclease I (NEB) and incubated the tube at 37°C for 15 min.

    Article Title: Y-chromosome evidence supports asymmetric dog introgression into eastern coyotes
    Article Snippet: For a subsample of males we amplified a 658 bp fragment of the last intron of the Zfy gene with published primers (LGL-331: Shaw et al. ; Yint-2-335: Wilson et al. ) as in Wilson et al. ( ). .. We purified polymerase chain reaction (PCR) products using Exosap-IT (USB Corporation, Cleveland, OH), or Exonuclease I and Antarctic Phosphatase (New England BioLabs Inc., Ipswich, MA), prior to sequencing on a MegaBACE 1000 (GE Healthcare) or an AB3730 (Applied Biosystems). .. We edited and aligned sequences in Bioedit (v7.0.9, Hall ) or MEGA (v5, Tamura et al. ).

    Article Title: Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity
    Article Snippet: The PCR products were treated with shrimp alkaline phosphatase (Roche Applied Science, Indianapolis, IN) and exonuclease I (NEB, Ipswich, MA), and then sequenced with the BigDye Terminator ver.1.1 (Applied Biosystems, Foster City, CA). .. The PCR products were treated with shrimp alkaline phosphatase (Roche Applied Science, Indianapolis, IN) and exonuclease I (NEB, Ipswich, MA), and then sequenced with the BigDye Terminator ver.1.1 (Applied Biosystems, Foster City, CA).

    Article Title: Magnetic resonance imaging and genetic investigation of a case of rottweiler leukoencephalomyelopathy
    Article Snippet: For the DARS2 mutation analysis, suitable PCR products were amplified using AmpliTaq Gold 360 (Life Technologies). .. The PCR products were resequenced after rAPid alkaline phosphatase (Roche) and exonuclease I (New England Biolabs) treatment using both PCR primers and the ABI BigDye Terminator Sequencing Kit 3.1 (Life Technologies) in an ABI 3730 sequencer (see Additional file : Table S1). .. The sequence data were analysed using Sequencer 4.9 software (GeneCodes).

    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
    Article Snippet: Transformants were transferred to Luria-Bertani (LB) broth containing kanamycin (50 μ g/mL) in 96-well tissue culture plates, incubated for 16 h at 37°C at 225 rpm, then screened with PCR using the M13f and M13r primers ( ) to check for the presence of an insert. .. Proper-sized transformants were purified with exonuclease 1 and antarctic phosphatase (New England BioLabs, Inc., Ipswich, MA) for 45 min at 37°C then at 95°C for 15 min prior to sequencing. .. Purified plasmid DNA was mixed with the M13f primer then submitted to the Cornell University Life Sciences Core Laboratories Center.

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: Alternatively, the PCR products were separated using an HDA-GT12 DNA analyzer and scored by Biocalculator software (both from eGene, Irvine, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs). .. DNA sequencing was performed using ABI BigDye Terminator (v3.1; Applied Biosystems, Foster City, CA) according to the manufacturer's protocol, except that 5-μl reactions were performed with 0.25 μl of BigDye on an ABI 3730xl DNA sequencing machine with 50 cm arrays.

    Article Title: A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3
    Article Snippet: All primer sequences can be found in Supporting Information, . .. PCR product clean-up was carried out using ExoI (NEB, Ipswich, MA, USA) and SAP (USB, part of Affymetrix, Santa Clara, CA, USA) with standard protocols, and sequencing reactions, using BigDye Terminator v3.1 Cycle Sequencing kits, were run on a 3730xl DNA Analyzer (Applied Biosystems). .. Base calling and quality assessment were carried out using the Contig Express programme from the Vector NTI package (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22-1 as an allele of chromatin- remodelling factor gene swr1
    Article Snippet: Genomic DNA was amplified by yeast colony polymerase chain reaction (PCR), using a blend of Pwo and Taq polymerase. .. The resulting 2 kb PCR products were treated with exonuclease I and Antarctic phosphatase (NEB) and directly used in sequencing reactions. .. Lasergene (DNAStar) was used to assemble and analyse the sequences.

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs. .. The sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacture's instructions.

    Injection:

    Article Title: Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis
    Article Snippet: Paragraph title: Microinjection of targeting oligonucleotides/Rad52 mixtures and DNA extraction from injected embryos ... To extract genomic DNA, individual embryos were placed into 25 μl of ice-cold DNA extraction buffer (10 mM Tris–HCl, pH 8.0, 2 mM EDTA, 0.2% Triton X-100, 200 μg/ml Proteinase K) and incubated at 50°C for 16 h, then at 95°C for 10 min. To remove remaining targeting oligonucleotide, 25 μl of Exonuclease I mixture [0.04 U/μl Exonuclease I (New England Biolabs), 134 mM Glycine-KOH, pH 9.5, 13.4 mM MgCl2 , 20 mM 2-mercaptoethanol] was added, and incubated at 37°C for 1 h and at 80°C for 20 min.

    Binding Assay:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
    Article Snippet: Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min). .. Distinct primer assays were generated by adding individual primer pairs (5μM) together with a mix of 2x Assay Loading Reagent (Fluidigm) and 1x DNA suspension buffer to each well of a new plate.

    Article Title: Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma
    Article Snippet: Following pre-amplification unincorporated primers were removed by exonuclease I digestion (2 μl of 4 U/μl exonuclease I (New England Biolabs Inc., #M0293S) by incubation in a thermal cycler at 37°C for 30 minutes followed by 80°C for 15 minutes. .. Following pre-amplification unincorporated primers were removed by exonuclease I digestion (2 μl of 4 U/μl exonuclease I (New England Biolabs Inc., #M0293S) by incubation in a thermal cycler at 37°C for 30 minutes followed by 80°C for 15 minutes.

    Article Title: Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization
    Article Snippet: Samples were treated with Exonuclease I (New England BioLabs, Ipswich, MA) to remove unincorporated primers by adding 2 μl (at 4 U/μl) to each STA reaction. .. Operation of instruments and handling and processing of the IFC controller HX were performed using standardized protocol for gene expression analysis provided by Fluidigm®.

    Staining:

    Article Title: Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma
    Article Snippet: Using the same staining protocol and gating strategy outlined above, 10 cells in triplicate were sorted from selected cTfh subsets of 5 normal subjects and 4 MZL patients. .. Following pre-amplification unincorporated primers were removed by exonuclease I digestion (2 μl of 4 U/μl exonuclease I (New England Biolabs Inc., #M0293S) by incubation in a thermal cycler at 37°C for 30 minutes followed by 80°C for 15 minutes.

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: PCR samples were stained prior to electrophoresis with 1× GelRed (Biotium, Hayward, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs).

    DNA Extraction:

    Article Title: Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity
    Article Snippet: DNA samples were extracted from peripheral blood using a DNA extraction kit (QiaAmp; Qiagen, Chatsworth, CA). .. The PCR products were treated with shrimp alkaline phosphatase (Roche Applied Science, Indianapolis, IN) and exonuclease I (NEB, Ipswich, MA), and then sequenced with the BigDye Terminator ver.1.1 (Applied Biosystems, Foster City, CA).

    Article Title: Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis
    Article Snippet: One-cell stage embryos were microinjected with 1–2 nl of the mixture, and cultured until shield to 80% epiboly stages. .. To extract genomic DNA, individual embryos were placed into 25 μl of ice-cold DNA extraction buffer (10 mM Tris–HCl, pH 8.0, 2 mM EDTA, 0.2% Triton X-100, 200 μg/ml Proteinase K) and incubated at 50°C for 16 h, then at 95°C for 10 min. To remove remaining targeting oligonucleotide, 25 μl of Exonuclease I mixture [0.04 U/μl Exonuclease I (New England Biolabs), 134 mM Glycine-KOH, pH 9.5, 13.4 mM MgCl2 , 20 mM 2-mercaptoethanol] was added, and incubated at 37°C for 1 h and at 80°C for 20 min. .. Mutant template was detected with a pair of PCR primers (BrulATRFO: 5′-AAGCTGAACAGTCAGAGTTGGTC-3′ (T m = 55°C) and BrulMutMcAsR: 5′-TGATTTCATAAACTGCACCC-3′ (T m = 48°C).

    In Vivo:

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
    Article Snippet: Paragraph title: In vivo TMP–DNA PB and selection of DNA-crosslinked fragments ... To select the crosslinked fragments, DNA samples were boiled for 2 min, chilled on ice and then digested with excess Exonuclease I (NE BioLabs) during 4 h. Undigested DNA was recovered with a GFX filtration kit (Amersham) in 50 µl of water and further digested with excess λ Exonuclease (NE BioLabs) during 6 h. The reaction terminated by inactivation of the enzyme at 80°C during 10 min. To examine TMP PB to purified yeast DNA, unreacted cells were disrupted using the Fast-Prep apparatus and total DNA was fragmented down to a 2 kb average size by using a Branson Sonifier, as above.

    Mutagenesis:

    Article Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
    Article Snippet: These regions contain a mutant count of greater than 1 for any single point mutation. .. Following multiplex cycling, 1 µl of exonuclease I (New England Biolabs, Ipswich, MA, USA) was added to each reaction and incubated at 37°C for 30 min and 80°C for 15 min to remove unincorporated primers.

    Article Title: Magnetic resonance imaging and genetic investigation of a case of rottweiler leukoencephalomyelopathy
    Article Snippet: For the DARS2 mutation analysis, suitable PCR products were amplified using AmpliTaq Gold 360 (Life Technologies). .. The PCR products were resequenced after rAPid alkaline phosphatase (Roche) and exonuclease I (New England Biolabs) treatment using both PCR primers and the ABI BigDye Terminator Sequencing Kit 3.1 (Life Technologies) in an ABI 3730 sequencer (see Additional file : Table S1).

    Isolation:

    Article Title: A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3
    Article Snippet: Sequencing was performed on DNA samples isolated from three of the most severely affected family members (II-3, III-4 and III-6). .. PCR product clean-up was carried out using ExoI (NEB, Ipswich, MA, USA) and SAP (USB, part of Affymetrix, Santa Clara, CA, USA) with standard protocols, and sequencing reactions, using BigDye Terminator v3.1 Cycle Sequencing kits, were run on a 3730xl DNA Analyzer (Applied Biosystems).

    Size-exclusion Chromatography:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
    Article Snippet: Next, pre-amplification mix, consisting of pooled mixture of all primer assays (500nM), 5× PreAmp Master Mix (Fluidigm) and H2 O, was added to each well and run on a thermocycler (95°C for 5 min followed by 18 cycles: 96°C for 5 sec 60°C for 6 min). .. Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min).

    Labeling:

    Article Title: Natural Genetic Variation of Integrin Alpha L (Itgal) Modulates Ischemic Brain Injury in Stroke
    Article Snippet: Amplicons containing coding SNPs were amplified by conventional PCR, and 15 ul of PCR products were treated with 1 U exonuclease I (New England Biolabs) and 5 U of Shrimp Alkaline Phosphatase (SAP) (Promega). .. Purified PCR products were used in combination with a conventional primer designed to sit at the nucleotide to the immediate 5′ position of a coding SNPs in the transcript.

    Purification:

    Article Title: Y-chromosome evidence supports asymmetric dog introgression into eastern coyotes
    Article Snippet: For a subsample of males we amplified a 658 bp fragment of the last intron of the Zfy gene with published primers (LGL-331: Shaw et al. ; Yint-2-335: Wilson et al. ) as in Wilson et al. ( ). .. We purified polymerase chain reaction (PCR) products using Exosap-IT (USB Corporation, Cleveland, OH), or Exonuclease I and Antarctic Phosphatase (New England BioLabs Inc., Ipswich, MA), prior to sequencing on a MegaBACE 1000 (GE Healthcare) or an AB3730 (Applied Biosystems). .. We edited and aligned sequences in Bioedit (v7.0.9, Hall ) or MEGA (v5, Tamura et al. ).

    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
    Article Snippet: Transformants were transferred to Luria-Bertani (LB) broth containing kanamycin (50 μ g/mL) in 96-well tissue culture plates, incubated for 16 h at 37°C at 225 rpm, then screened with PCR using the M13f and M13r primers ( ) to check for the presence of an insert. .. Proper-sized transformants were purified with exonuclease 1 and antarctic phosphatase (New England BioLabs, Inc., Ipswich, MA) for 45 min at 37°C then at 95°C for 15 min prior to sequencing. .. Purified plasmid DNA was mixed with the M13f primer then submitted to the Cornell University Life Sciences Core Laboratories Center.

    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction
    Article Snippet: This was then followed by 68°C for 5 min. After PCR reactions, 0.5 μl of exonuclease I (10 U, from either New England Biolabs or Epicentre) was added per 50 μl of PCR products and incubated at 37°C for 1 h followed by heat inactivation at 80°C for 20 min. .. This was then followed by 68°C for 5 min. After PCR reactions, 0.5 μl of exonuclease I (10 U, from either New England Biolabs or Epicentre) was added per 50 μl of PCR products and incubated at 37°C for 1 h followed by heat inactivation at 80°C for 20 min.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Circularized MIPs were dissociated from the cDNA by holding the sample at 95°C for 5 minutes and chilled on ice. .. The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes.

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
    Article Snippet: DNA was precipitated with EtOH, washed with 70% EtOH and resuspended in 100 µl of water. .. To select the crosslinked fragments, DNA samples were boiled for 2 min, chilled on ice and then digested with excess Exonuclease I (NE BioLabs) during 4 h. Undigested DNA was recovered with a GFX filtration kit (Amersham) in 50 µl of water and further digested with excess λ Exonuclease (NE BioLabs) during 6 h. The reaction terminated by inactivation of the enzyme at 80°C during 10 min. To examine TMP PB to purified yeast DNA, unreacted cells were disrupted using the Fast-Prep apparatus and total DNA was fragmented down to a 2 kb average size by using a Branson Sonifier, as above. .. Following proteinase K and RNAse I incubations, DNA fragments were extracted twice with phenol and once with phenol–chloroform, precipitated with EtOH, washed with 70% EtOH and resuspended in 100 µl of water.

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: Approximate size of the PCR amplicons was determined by electrophoresis on 1.5% agarose gel with a DNA ladder (GeneRuler™ DNA Ladder Mix, Fermentas). .. PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs. .. The sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacture's instructions.

    Polymerase Chain Reaction:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
    Article Snippet: Similar to before [ ], PCR plates were thawed and pre-heated for 90 seconds at 65°C. .. Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min).

    Article Title: Natural Genetic Variation of Integrin Alpha L (Itgal) Modulates Ischemic Brain Injury in Stroke
    Article Snippet: PCR was performed using an appropriate dilution of cDNA generated from the cerebral cortex of F1 (B6×BALB) animals. .. Amplicons containing coding SNPs were amplified by conventional PCR, and 15 ul of PCR products were treated with 1 U exonuclease I (New England Biolabs) and 5 U of Shrimp Alkaline Phosphatase (SAP) (Promega). .. Purified PCR products were used in combination with a conventional primer designed to sit at the nucleotide to the immediate 5′ position of a coding SNPs in the transcript.

    Article Title: Y-chromosome evidence supports asymmetric dog introgression into eastern coyotes
    Article Snippet: For a subsample of males we amplified a 658 bp fragment of the last intron of the Zfy gene with published primers (LGL-331: Shaw et al. ; Yint-2-335: Wilson et al. ) as in Wilson et al. ( ). .. We purified polymerase chain reaction (PCR) products using Exosap-IT (USB Corporation, Cleveland, OH), or Exonuclease I and Antarctic Phosphatase (New England BioLabs Inc., Ipswich, MA), prior to sequencing on a MegaBACE 1000 (GE Healthcare) or an AB3730 (Applied Biosystems). .. We edited and aligned sequences in Bioedit (v7.0.9, Hall ) or MEGA (v5, Tamura et al. ).

    Article Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
    Article Snippet: Multiplex PCR cycling was performed according to the manufacturer’s recommendations (Kapa Biosystems) for a total of 15–25 PCR cycles using 63°C as optimal annealing temperature. .. Following multiplex cycling, 1 µl of exonuclease I (New England Biolabs, Ipswich, MA, USA) was added to each reaction and incubated at 37°C for 30 min and 80°C for 15 min to remove unincorporated primers.

    Article Title: Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity
    Article Snippet: Polymerase chain reaction (PCR) was performed with optimized annealing temperatures. .. The PCR products were treated with shrimp alkaline phosphatase (Roche Applied Science, Indianapolis, IN) and exonuclease I (NEB, Ipswich, MA), and then sequenced with the BigDye Terminator ver.1.1 (Applied Biosystems, Foster City, CA). .. The samples were denatured and analyzed with a DNA sequencer (3100 or 3730 Genetic Analyzer; Applied Biosystems).

    Article Title: Magnetic resonance imaging and genetic investigation of a case of rottweiler leukoencephalomyelopathy
    Article Snippet: For the DARS2 mutation analysis, suitable PCR products were amplified using AmpliTaq Gold 360 (Life Technologies). .. The PCR products were resequenced after rAPid alkaline phosphatase (Roche) and exonuclease I (New England Biolabs) treatment using both PCR primers and the ABI BigDye Terminator Sequencing Kit 3.1 (Life Technologies) in an ABI 3730 sequencer (see Additional file : Table S1). .. The sequence data were analysed using Sequencer 4.9 software (GeneCodes).

    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
    Article Snippet: Transformants were transferred to Luria-Bertani (LB) broth containing kanamycin (50 μ g/mL) in 96-well tissue culture plates, incubated for 16 h at 37°C at 225 rpm, then screened with PCR using the M13f and M13r primers ( ) to check for the presence of an insert. .. Proper-sized transformants were purified with exonuclease 1 and antarctic phosphatase (New England BioLabs, Inc., Ipswich, MA) for 45 min at 37°C then at 95°C for 15 min prior to sequencing.

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: Synthesized universal fragments were gel-purified (QIAquick Gel Extraction Kit, Qiagen). .. Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes. .. To obtain the libraries of DNA templates two universal fragments (1, 2 or 3, 4, ) were combined with one of the 31 variable PCR fragments using OE-PCR.

    Article Title: Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization
    Article Snippet: The PCR plate was transferred to a thermal cycler and subjected to the following thermal protocol: 95°C, 10 min; 14 cycles (95°C ,15 s; 60°C, 4 min); 4°C hold. .. Samples were treated with Exonuclease I (New England BioLabs, Ipswich, MA) to remove unincorporated primers by adding 2 μl (at 4 U/μl) to each STA reaction.

    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction
    Article Snippet: PCR was performed using PTC-225 PCR machine (Peltier Thermal Cycler) with the following settings: 94°C for 4 min, this was followed by 35 cycles of 94°C for 30 s, 60°C for 30 s and 68°C for 1 min (Bsd ) or 2–3 min (Neo and retrieval backbone). .. This was then followed by 68°C for 5 min. After PCR reactions, 0.5 μl of exonuclease I (10 U, from either New England Biolabs or Epicentre) was added per 50 μl of PCR products and incubated at 37°C for 1 h followed by heat inactivation at 80°C for 20 min. .. The PCR products were then purified using Qiagen mini-preparation columns and eluted in 50 μl of PCR-grade water.

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: Alternatively, the PCR products were separated using an HDA-GT12 DNA analyzer and scored by Biocalculator software (both from eGene, Irvine, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs). .. DNA sequencing was performed using ABI BigDye Terminator (v3.1; Applied Biosystems, Foster City, CA) according to the manufacturer's protocol, except that 5-μl reactions were performed with 0.25 μl of BigDye on an ABI 3730xl DNA sequencing machine with 50 cm arrays.

    Article Title: A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3
    Article Snippet: All primer sequences can be found in Supporting Information, . .. PCR product clean-up was carried out using ExoI (NEB, Ipswich, MA, USA) and SAP (USB, part of Affymetrix, Santa Clara, CA, USA) with standard protocols, and sequencing reactions, using BigDye Terminator v3.1 Cycle Sequencing kits, were run on a 3730xl DNA Analyzer (Applied Biosystems). .. Base calling and quality assessment were carried out using the Contig Express programme from the Vector NTI package (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Article Title: Improved tools for efficient mapping of fission yeast genes: identification of microtubule nucleation modifier mod22-1 as an allele of chromatin- remodelling factor gene swr1
    Article Snippet: Genomic DNA was amplified by yeast colony polymerase chain reaction (PCR), using a blend of Pwo and Taq polymerase. .. The resulting 2 kb PCR products were treated with exonuclease I and Antarctic phosphatase (NEB) and directly used in sequencing reactions. .. Lasergene (DNAStar) was used to assemble and analyse the sequences.

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: Approximate size of the PCR amplicons was determined by electrophoresis on 1.5% agarose gel with a DNA ladder (GeneRuler™ DNA Ladder Mix, Fermentas). .. PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs. .. The sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacture's instructions.

    Quantitative RT-PCR:

    Article Title: Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma
    Article Snippet: Paragraph title: Microfluidic RT-qPCR ... Following pre-amplification unincorporated primers were removed by exonuclease I digestion (2 μl of 4 U/μl exonuclease I (New England Biolabs Inc., #M0293S) by incubation in a thermal cycler at 37°C for 30 minutes followed by 80°C for 15 minutes.

    FACS:

    Article Title: Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease
    Article Snippet: After FACS sorting, PCR plates were frozen and kept in -80°C until usage. .. Exonuclease mixture, containing of Exonuclease I (New England BioLabs), 10× Exonuclease I Reaction Buffer (New England BioLabs) and H2 O was added to each well to remove excessive primers and the plate was run on a thermocycler (37°C for 30 min, 80°C for 15 min).

    Activated Clotting Time Assay:

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
    Article Snippet: We suspended the collected beads in 50 μl of 1× Phusion HF buffer containing 0.25 mM dNTPs, 2 U Phusion Hot Start High-Fidelity DNA Polymerase (Finnzyme) and 0.8 μM Primer-3 (5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-3′). .. We transferred the supernatant to a new tube, added 1 μl of 20 U/μl Exonuclease I (NEB) and incubated the tube at 37°C for 15 min.

    Helicase-dependent Amplification:

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: Alternatively, the PCR products were separated using an HDA-GT12 DNA analyzer and scored by Biocalculator software (both from eGene, Irvine, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs).

    Plasmid Preparation:

    Article Title: Soil pathogen communities associated with native and non-native Phragmites australis populations in freshwater wetlands
    Article Snippet: PCR products from rhizosphere soils were cloned using INVα F' competent cells and the pCR®2.1 vector from the TA Cloning® Kit (Life Technologies, Grand Island, NY) according to the manufacturer's directions. .. Proper-sized transformants were purified with exonuclease 1 and antarctic phosphatase (New England BioLabs, Inc., Ipswich, MA) for 45 min at 37°C then at 95°C for 15 min prior to sequencing.

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: In addition, the fragment encoding for PP1 phosphatase catalytic subunit (GeneBank ID NP_002699) was amplified from PP1-encoding plasmid with primers 9176 and 9177 using PCR conditions as given for genomic PCR. .. Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes.

    Software:

    Article Title: Natural Genetic Variation of Integrin Alpha L (Itgal) Modulates Ischemic Brain Injury in Stroke
    Article Snippet: Amplicons containing coding SNPs were amplified by conventional PCR, and 15 ul of PCR products were treated with 1 U exonuclease I (New England Biolabs) and 5 U of Shrimp Alkaline Phosphatase (SAP) (Promega). .. Cycling conditions were as follows: 40 cycles of 95C° for 10 s, 50C° for 5 s, and 60C° for 30 s. The primer is then labeled with a fluorescently tagged dideoxynucleotide through a single base pair extension (SNaPshot kit from ABI).

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: Alternatively, the PCR products were separated using an HDA-GT12 DNA analyzer and scored by Biocalculator software (both from eGene, Irvine, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs).

    Irradiation:

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
    Article Snippet: To select the crosslinked fragments, DNA samples were boiled for 2 min, chilled on ice and then digested with excess Exonuclease I (NE BioLabs) during 4 h. Undigested DNA was recovered with a GFX filtration kit (Amersham) in 50 µl of water and further digested with excess λ Exonuclease (NE BioLabs) during 6 h. The reaction terminated by inactivation of the enzyme at 80°C during 10 min. To examine TMP PB to purified yeast DNA, unreacted cells were disrupted using the Fast-Prep apparatus and total DNA was fragmented down to a 2 kb average size by using a Branson Sonifier, as above. .. To select the crosslinked fragments, DNA samples were boiled for 2 min, chilled on ice and then digested with excess Exonuclease I (NE BioLabs) during 4 h. Undigested DNA was recovered with a GFX filtration kit (Amersham) in 50 µl of water and further digested with excess λ Exonuclease (NE BioLabs) during 6 h. The reaction terminated by inactivation of the enzyme at 80°C during 10 min. To examine TMP PB to purified yeast DNA, unreacted cells were disrupted using the Fast-Prep apparatus and total DNA was fragmented down to a 2 kb average size by using a Branson Sonifier, as above.

    Multiplex Assay:

    Article Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
    Article Snippet: Amplicon lengths ranged from 120 to 190 bp in size depending on the amplicon ( Supplementary Table S2 ). .. Following multiplex cycling, 1 µl of exonuclease I (New England Biolabs, Ipswich, MA, USA) was added to each reaction and incubated at 37°C for 30 min and 80°C for 15 min to remove unincorporated primers. .. Probe design and preparation .

    Selection:

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
    Article Snippet: We transferred the supernatant to a new tube, added 1 μl of 20 U/μl Exonuclease I (NEB) and incubated the tube at 37°C for 15 min. .. Following incubation at 70°C for 10 min to inactivate Exonuclease I, we purified the double-stranded template DNAs with 50 μl of AMPure XP twice as described above, except for reducing the volume of final elution to 20 μl.

    Article Title: A recombineering based approach for high-throughput conditional knockout targeting vector construction
    Article Snippet: The restriction maps of the selection cassettes and PL611 are depicted in Supplementary Figures 2–6. .. This was then followed by 68°C for 5 min. After PCR reactions, 0.5 μl of exonuclease I (10 U, from either New England Biolabs or Epicentre) was added per 50 μl of PCR products and incubated at 37°C for 1 h followed by heat inactivation at 80°C for 20 min.

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
    Article Snippet: Paragraph title: In vivo TMP–DNA PB and selection of DNA-crosslinked fragments ... To select the crosslinked fragments, DNA samples were boiled for 2 min, chilled on ice and then digested with excess Exonuclease I (NE BioLabs) during 4 h. Undigested DNA was recovered with a GFX filtration kit (Amersham) in 50 µl of water and further digested with excess λ Exonuclease (NE BioLabs) during 6 h. The reaction terminated by inactivation of the enzyme at 80°C during 10 min. To examine TMP PB to purified yeast DNA, unreacted cells were disrupted using the Fast-Prep apparatus and total DNA was fragmented down to a 2 kb average size by using a Branson Sonifier, as above.

    Agarose Gel Electrophoresis:

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: Approximate size of the PCR amplicons was determined by electrophoresis on 1.5% agarose gel with a DNA ladder (GeneRuler™ DNA Ladder Mix, Fermentas). .. PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs.

    Electrophoresis:

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: PCR samples were stained prior to electrophoresis with 1× GelRed (Biotium, Hayward, CA). .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs).

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: Approximate size of the PCR amplicons was determined by electrophoresis on 1.5% agarose gel with a DNA ladder (GeneRuler™ DNA Ladder Mix, Fermentas). .. PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs.

    Ethanol Precipitation:

    Article Title: Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)
    Article Snippet: PCR products were purified using exonuclease I (New England Biolabs) and shrimp alkaline phosphatase (Roche) and directly sequenced in both forward and reverse directions with the same primers as those used in the PCRs. .. The sequencing reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to manufacture's instructions.

    Concentration Assay:

    Article Title: Differential strand separation at critical temperature: A minimally disruptive enrichment method for low-abundance unknown DNA mutations
    Article Snippet: Multiplex pre-amplification from ∼20 ng genomic DNA or 10 ng plasma-circulating DNA was performed in a total volume of 25 µl using a mixture of 100 primers (50 paired sets) at a final concentration of 0.3 µM for each primer with 0.3 mM dNTPs, 3 mM MgCl2 , 1× Kapa HiFi buffer and 0.5 U of Kapa HiFi HotStart DNA polymerase (Kapa Biosystems, Woburn, MA, USA) reported to have an error rate of 2.8 × 10−7 mis-incorporations/bp. .. Following multiplex cycling, 1 µl of exonuclease I (New England Biolabs, Ipswich, MA, USA) was added to each reaction and incubated at 37°C for 30 min and 80°C for 15 min to remove unincorporated primers.

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: Primers 8817–8878 at 300 nM concentration were used in amplification of the genes 1–31 with 35 cycles. .. Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes.

    Article Title: Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1
    Article Snippet: The polymerase chain reaction (PCR) was performed in a 20 μl volume containing 10 ng of genomic DNA as a template, 200 μmol/L of each dNTP, 1× Standard Taq PCR buffer with 1.5 mmol/L MgCl2 , 1.2 U Taq polymerase (all from New England Biolabs, Ipswich, MA), and forward and reverse primers at a concentration of 0.25 μmol/L each. .. If sequencing was needed, PCR products were first treated with Exonuclease I and subsequently with Antarctic Phosphatase (both from New England Biolabs).

    Fractionation:

    Article Title: Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging
    Article Snippet: Paragraph title: Elution and size fractionation ... We transferred the supernatant to a new tube, added 1 μl of 20 U/μl Exonuclease I (NEB) and incubated the tube at 37°C for 15 min.

    Migration:

    Article Title: Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma
    Article Snippet: A custom panel of 96 genes (including 4 candidate reference genes) implicated in CD4+ T-cell biology (migration, differentiation, effector function and cell signalling pathways) was created, and primers designed using the D3 Assay Design service (Fluidigm, ) ( ). .. Following pre-amplification unincorporated primers were removed by exonuclease I digestion (2 μl of 4 U/μl exonuclease I (New England Biolabs Inc., #M0293S) by incubation in a thermal cycler at 37°C for 30 minutes followed by 80°C for 15 minutes.

    Gel Extraction:

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System
    Article Snippet: Synthesized universal fragments were gel-purified (QIAquick Gel Extraction Kit, Qiagen). .. Variable ORF-encoded fragments were freed from residual primers by treatment with 15 U/ml Exonuclease I (NEB), which was added directly to the final PCR reaction mixture, for 30 minutes at 37°C followed by nuclease inactivation at 85°C for 30 minutes.

    Variant Assay:

    Article Title: Genetic variants of FZD4 and LRP5 genes in patients with advanced retinopathy of prematurity
    Article Snippet: The PCR products were treated with shrimp alkaline phosphatase (Roche Applied Science, Indianapolis, IN) and exonuclease I (NEB, Ipswich, MA), and then sequenced with the BigDye Terminator ver.1.1 (Applied Biosystems, Foster City, CA). .. Each sequence change was evaluated by two examiners using the Phred/Phrap/Consed program [ ].

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    New England Biolabs e coli exoi
    <t>DNA</t> with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli <t>ExoI.</t> The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    E Coli Exoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs λ exonuclease buffer
    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of <t>λ-exonuclease</t> (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .
    λ Exonuclease Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Hybridization, Nucleic Acid Electrophoresis, Electrophoresis, Blocking Assay, Incubation, Labeling

    Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk  with 1 pmol  miD156s  at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk  with 1 pmol  miD156s  at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk  with 1 pmol  miD156s  at different temperatures by traditional Northern hybridization.  miD156  marked “+”,  miD156  with one-base mismatch marked “−1”,  miD156  with three-base mismatch marked “−3” and  miD156  with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of  miD156 ; 10f represents 10 fmol of  miD156 ; 5f represents 5 fmol of  miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Northern Blot, Hybridization

    Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Cdc13 protects the telomere 5′ end when bound 3 nt away from the ds-ss junction. ( a ) D10S16 contains 10 bp of telomere dsDNA and a 16 nt ssDNA 3′ overhang. The Cdc13 MBS (bold text) is located 3 nt from the ds-ss junction. The black bar represents the 14 bp guide sequence (5′-GTCACACGTCACAC-3′) used for ensuring proper annealing. “*” Indicates the radioactive label at the 3′ end of the C-strand, used for detecting the substrate and its degradation products. ( b ) Sequencing gel with the 5′DEPA reaction products. D10S16 was either pre-bound by Cdc13 or incubated with non-DNA binding BSA protein. An aliquot was taken out before addition of λ-exonuclease (-), then λ-exonuclease was added and aliquots of the reactions were stopped at different time points (20; 40; 60; 120; 240 s). ( c ) Graph showing the quantification of the gel shown in ( b ). The amount of uncleaved substrate (S) relative the reaction start point was calculated by measuring the volume of the upper two uncleaved substrate bands normalized to the volume of the loading control band (LC). Reaction products are denoted next to the gel (P). The uncropped gel is presented in Supplementary Fig. S2 .

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Binding Assay, Liquid Chromatography

    Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: Schematic figure summarizing the results of this work and how it is proposed to relate to different in vivo situations. ( a ) Shows the different substrate tested with Cdc13 or Rap1 pre-bound at their respective MBS at various distances relative the ds-ss junction. “ + ” indicates protection, while “−’’ indicates no protection. ( b ) Protection by Rap1 when the 3′ overhang is very short and unable to accommodate Cdc13 binding. ( c ) Protection by Rap1 in a hypothetical situation where Cdc13 is bound very far away from the ds-ss junction (longer than tested here). ( d ) Protection may be provided by Cdc13 alone when the 3′ overhang accommodates its binding. ( e ) The wild type Rap1 DBD 337–582 is firmly attached to its MBS, and fully protects the 5′ end from degradation by λ-exonuclease. ( f ) The Rap1 wrapping loop mutant DBD 337–556 is only partly attached to the MBS, leaving the 5′ end accessible to λ-exonuclease, which cleaves off the first 3 nt of DNA before being halted at a site where the mutant DBD is more firmly attached.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: In Vivo, Binding Assay, Mutagenesis

    ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Journal: Scientific Reports

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends

    doi: 10.1038/s41598-017-08663-x

    Figure Lengend Snippet: ( a ) Schematic illustration of the 5′ DNA end protection assay (DEPA). DNA oligonucleotides are annealed to form model telomeres with a double stranded part and a single stranded 3′ overhang (I). All oligonucleotides contain a short non-telomeric guide sequence to ensure efficient annealing while the telomere part is varied to create different length overhangs and different 5′ permutations. λ-exonuclease selectively cleaves the 5′ phosphorylated end (II) of the shorter C-strand oligonucleotide which is 3′ end labelled (*). The reaction progresses in the 5′ → 3′ direction (II). To assay for 5′ end protection, Cdc13 is pre-bound to the telomere end before adding λ-exonuclease to the reaction, which will inhibit the exonuclease (III). ( b ) Schematic illustration of the assay read out. Reactions are stopped at different incubation times, de-proteinized, ethanol precipitated and run on a 10% denaturing polyacrylamide sequencing gel. A labelled oligonucleotide loading control (LC) is added before ethanol precipitation which migrates above the 3′ labelled C-strand substrate (S) on the gel. As the exonuclease reaction progresses, products of decreasing size (P) appears on the gel while the uncleaved substrate (S) diminishes. Lane I, no enzyme control (0 s); lane IIa, shorter incubation time; lane IIb, longer incubation time; lane III, a reaction where the substrate was pre-incubated with Cdc13 which gave full protection.

    Article Snippet: For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction.

    Techniques: Sequencing, Incubation, Liquid Chromatography, Ethanol Precipitation