Structured Review

TaKaRa exonuclease iii
<t>Exosomes</t> secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from <t>three</t> independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.
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Images

1) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Journal: Nature Communications

doi: 10.1038/ncomms15287

Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.
Figure Legend Snippet: Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

Techniques Used: Transfection, Western Blot

Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
Figure Legend Snippet: Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

Techniques Used: Inhibition, Mouse Assay, Injection, Plasmid Preparation, Luciferase, shRNA, Transfection, Imaging, Western Blot, Isolation, Immunofluorescence, Staining

Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
Figure Legend Snippet: Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

Techniques Used: Transfection, Infection, Recombinant, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Titration, Generated

Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
Figure Legend Snippet: Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

Techniques Used: Inhibition, Transfection, Western Blot, Isolation, Cell Culture, Immunofluorescence, Staining, Infection, Plasmid Preparation, Selection

Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P
Figure Legend Snippet: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

Techniques Used: Over Expression, Infection, Plasmid Preparation, Selection, Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing

Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P
Figure Legend Snippet: Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

Techniques Used: Transfection, Western Blot, Immunofluorescence, Staining

2) Product Images from "A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors"

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0047159

Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p
Figure Legend Snippet: Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Fluorescence

Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p
Figure Legend Snippet: Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p

Techniques Used: Injection, Plasmid Preparation, Mouse Assay

3) Product Images from "RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection"

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

Journal: Scientific Reports

doi: 10.1038/s41598-018-26132-x

Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.
Figure Legend Snippet: Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.

Techniques Used: In Vitro, Ligation, Sequencing, Marker, Concentration Assay, Electrophoresis

4) Product Images from "Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system"

Article Title: Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system

Journal:

doi: 10.1093/nar/gnh039

The MALDI-TOF MS spectra for genotyping of SNP in apoE gene by using exonuclease III/nuclease S1/PNA systems. ( A ) Analysis of dsDNA from apoE 4 (250 bp) at codon 112 using PNA (S112G) . Reaction conditions: [ apoE DNA] = 3 µM,
Figure Legend Snippet: The MALDI-TOF MS spectra for genotyping of SNP in apoE gene by using exonuclease III/nuclease S1/PNA systems. ( A ) Analysis of dsDNA from apoE 4 (250 bp) at codon 112 using PNA (S112G) . Reaction conditions: [ apoE DNA] = 3 µM,

Techniques Used: Mass Spectrometry

5) Product Images from "Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes"

Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes

Journal: Particle and Fibre Toxicology

doi: 10.1186/1743-8977-8-1

Effects of endocytosis and NADPH oxidase inhibitor on DNA damage by silica particle treatment . Effects of endocytosis inhibitor ( A and B ) or NADPH oxidase inhibitor ( C and D ) on DNA strand breaks induced by silica particle treatment in HaCaT cells. ( A and B ) HaCaT cells were pretreated with 10 mM cytochalasin D (Cyto D) for 30 min (Cyto D + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. ( C and D ) HaCaT cells were pretreated with 40 mM apocynin (Apo) for 30 min (Apo + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. As a positive control, HaCaT cells were treated with 0.2 mM H 2 O 2 for 3 h. ( A and C ) Column height shows the tail length. ( B and D ) Column height shows the tail moment. Data shown are means ± SD of at least 16 cells per sample. Results shown are representative of more than three independent experiments. * P
Figure Legend Snippet: Effects of endocytosis and NADPH oxidase inhibitor on DNA damage by silica particle treatment . Effects of endocytosis inhibitor ( A and B ) or NADPH oxidase inhibitor ( C and D ) on DNA strand breaks induced by silica particle treatment in HaCaT cells. ( A and B ) HaCaT cells were pretreated with 10 mM cytochalasin D (Cyto D) for 30 min (Cyto D + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. ( C and D ) HaCaT cells were pretreated with 40 mM apocynin (Apo) for 30 min (Apo + nSP70) or nSP70 alone, prior to incubation with 90 mg/ml nSP70 for 3 h. As a positive control, HaCaT cells were treated with 0.2 mM H 2 O 2 for 3 h. ( A and C ) Column height shows the tail length. ( B and D ) Column height shows the tail moment. Data shown are means ± SD of at least 16 cells per sample. Results shown are representative of more than three independent experiments. * P

Techniques Used: Incubation, Positive Control

6) Product Images from "In Vitro Reconstitution of the End Replication Problem"

Article Title: In Vitro Reconstitution of the End Replication Problem

Journal:

doi: 10.1128/MCB.21.17.5753-5766.2001

Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ-32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
Figure Legend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ-32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.

Techniques Used: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay

Related Articles

Clone Assay:

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The PCR product was digested by Sma I and Bgl II , and inserted into the cloning site of pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA). .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al . .. [ ], and S1-nuclease (TaKaRa Bio) at 23°C for 15 min.

Amplification:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: Amplification of eGFP -MiLV was performed by PCR under the following conditions: initial 94°C denaturation (2 min), followed by 35 cycles of three PCR steps (each cycle: 30 s at 94°C, 30 s at 54°C and 80 s at 72°C), and terminated with an extension prolongation for 5 min at 72°C. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: A 1.43 kb DNA upstream fragment of S100A9 promoter [ ] was amplified by PCR according to a modified method of Kerkhoff et al. [ ]. .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al .

Positive Control:

Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes
Article Snippet: After 24 h, cells were treated with various concentrations of nSP70, nSP300, mSP1000, 0.2 mM H2 O2 (positive control) or PBS (negative control). .. Ten μg of DNA was converted to single stranded DNA by incubation with 180 U Exonuclease III (Takara Biotech., Japan) at 37°C for 1 h. The DNA was heated at 95°C for 5 min, rapidly chilled on ice, and digested to nucleosides by incubation with 0.6 U nuclease P1 (Takara) at 37°C for 1 h followed by treatment with 0.6 U E. coli alkaline phosphatase (Takara) for a further 1 h. The reaction mixture was centrifuged (6000 × g for 1 min) and the supernatant used for the 8-OHdG assay.

Blocking Assay:

Article Title: Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness
Article Snippet: DNA extracted from these cultures was resuspended in 30 μ l TE buffer (pH, 7.4) and quantified on a NanoDrop device (Thermo Scientific); typically, 4–5 μ g/ml. .. Exonuclease III buffer (10 μl, 10×) and 1000 units of Exonuclease III (Takara Biotechnology, Dalian, China) were added, plus distilled water to 100 μ l, followed by incubation at 37°C in a heat block for 48 hours. .. From the digest, 20 μ l was mixed vigorously with 60 μ l of Acetonitrile (Fisher Scientific, Loughborough, UK) and centrifuged (13,000× g , 5 minutes).

Electrophoresis:

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). .. Exonuclease III treatment was performed for 1.5 or 3 min at 15°C, and λ exonuclease treatment was performed for 1 or 2 min at 15°C.

Modification:

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: CO-FISH assay was performed based on the method described , with minor modification. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: CO-FISH assay was performed as described previously , with minor modification. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: A 1.43 kb DNA upstream fragment of S100A9 promoter [ ] was amplified by PCR according to a modified method of Kerkhoff et al. [ ]. .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al .

Incubation:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: Then, the hybridization mixture was incubated at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes
Article Snippet: After 3 h, cellular DNA was isolated using DNeasy tissue kit (QIAGEN, Germany). .. Ten μg of DNA was converted to single stranded DNA by incubation with 180 U Exonuclease III (Takara Biotech., Japan) at 37°C for 1 h. The DNA was heated at 95°C for 5 min, rapidly chilled on ice, and digested to nucleosides by incubation with 0.6 U nuclease P1 (Takara) at 37°C for 1 h followed by treatment with 0.6 U E. coli alkaline phosphatase (Takara) for a further 1 h. The reaction mixture was centrifuged (6000 × g for 1 min) and the supernatant used for the 8-OHdG assay. .. The amount of 8-OHdG was measured according to the protocol of the competitive ELISA kit (8-OHdG check; Japan Institute for the Control of Aging, Japan).

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: Subconfluent ES cells were incubated with BrdU (10 μM) for 10-12 h. Nocodazole was added to enrich metaphase 2 h prior to cell harvest, and metaphase spreads were prepared as described above for telomere QFISH. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: Chromosome slides were treated with RNase A, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml), incubated in 2 × SSC (Invitrogen) for 15 min and exposed to 365 nm UV light (Stratalinker 1800UV irradiator) for 40 min. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness
Article Snippet: DNA extracted from these cultures was resuspended in 30 μ l TE buffer (pH, 7.4) and quantified on a NanoDrop device (Thermo Scientific); typically, 4–5 μ g/ml. .. Exonuclease III buffer (10 μl, 10×) and 1000 units of Exonuclease III (Takara Biotechnology, Dalian, China) were added, plus distilled water to 100 μ l, followed by incubation at 37°C in a heat block for 48 hours. .. From the digest, 20 μ l was mixed vigorously with 60 μ l of Acetonitrile (Fisher Scientific, Loughborough, UK) and centrifuged (13,000× g , 5 minutes).

Luciferase:

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The PCR product was digested by Sma I and Bgl II , and inserted into the cloning site of pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA). .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al .

Expressing:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: Then, 1 µl ligation mixture was used as the template to amplify eGFP - MiLV by PCR by use of a single primer (5′ ACA AGT TCA GCG TGT CCG 3′, annealing to 751 to 768 of the eGFP expression cassette) in a 50 µl reaction system. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Acrylamide Gel Assay:

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). .. Exonuclease III treatment was performed for 1.5 or 3 min at 15°C, and λ exonuclease treatment was performed for 1 or 2 min at 15°C.

Hybridization:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: Then, the hybridization mixture was incubated at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Transfection:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Ligation:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: Then, 1 µl ligation mixture was used as the template to amplify eGFP - MiLV by PCR by use of a single primer (5′ ACA AGT TCA GCG TGT CCG 3′, annealing to 751 to 768 of the eGFP expression cassette) in a 50 µl reaction system. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: Then, the hybridization mixture was incubated at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al . .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al .

Cell Culture:

Article Title: Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness
Article Snippet: T. brucei bloodstream forms were incubated with 100 μ M of 5-fluoro-2′-deoxyuridine or 5-fluorouracil (12 hours, 37°C, 5% CO2 ); untreated control cells were cultured in parallel. .. Exonuclease III buffer (10 μl, 10×) and 1000 units of Exonuclease III (Takara Biotechnology, Dalian, China) were added, plus distilled water to 100 μ l, followed by incubation at 37°C in a heat block for 48 hours.

Article Title: Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation
Article Snippet: Cell samples from different days (OSKM/OSKMZ) were cultured as previously described . .. The BrdU-substituted DNA was digested with 3 U/μl Exonuclease III (Takara) for 10 min at room temperature.

DNA Sequencing:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Sequencing:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: To prepare the hybridization mixture, 25 pmol of the ribonucleotide (5′-rGrCrGrArUrCrArCrArUrGrArUrCrUrArCrurUrCrGrGrCrUrUrCrGrUrGrA-3′, 30-mer) and 100 pmol of the padlock probe for testing ligation efficiency (5′Pho-AGATCATGTGATCGCgaattcgccagggttttcccagtcacgactTCACGAAGCCGAAGT-3′, 60-mer, uppercase letters are homologous to the ribonucleotide sequence) were added to a mixture containing 20 mM Tris-acetate (pH 7.5), 50 mM potassium glutamate (KGlu), and 0.1 mM EDTA in a final volume of 10 µl. .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA).

Injection:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Imaging:

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). .. DNA fragments were further digested with restriction enzymes and separated by 6% denaturing acrylamide gel electrophoresis.

DNA Extraction:

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells
Article Snippet: The size distribution and concentration of the exosomes were determined using a NanoSight LM10 system (NanoSight Ltd.). .. To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions . .. After heat inactivation, the exosomal DNA was purified by Proteinase K (Wako) treatment.

Nucleic Acid Electrophoresis:

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: Paragraph title: Analysis of replication products by denaturing gel electrophoresis. ... The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).

Radioactivity:

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). .. DNA fragments were further digested with restriction enzymes and separated by 6% denaturing acrylamide gel electrophoresis.

Fluorescence:

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: Paragraph title: Chromosome orientation fluorescence in situ hybridization ... The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Isolation:

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells
Article Snippet: Paragraph title: Quantitative measurement of isolated exosomal DNA ... To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes
Article Snippet: After 3 h, cellular DNA was isolated using DNeasy tissue kit (QIAGEN, Germany). .. Ten μg of DNA was converted to single stranded DNA by incubation with 180 U Exonuclease III (Takara Biotech., Japan) at 37°C for 1 h. The DNA was heated at 95°C for 5 min, rapidly chilled on ice, and digested to nucleosides by incubation with 0.6 U nuclease P1 (Takara) at 37°C for 1 h followed by treatment with 0.6 U E. coli alkaline phosphatase (Takara) for a further 1 h. The reaction mixture was centrifuged (6000 × g for 1 min) and the supernatant used for the 8-OHdG assay.

Microscopy:

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: The BrdU-substituted DNA was digested with Exonuclease III (Takara). .. Slides were hybridized, washed, dehydrated, and counterstained with VectaShield antifade medium (Vector), containing 1.25 μg/ml DAPI.

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: The BrdU-substituted DNA was digested with Exonuclease III (Takara). .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Purification:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: Products obtained from in vitro replication performed in the presence of [α-32 P]dATP were purified as outlined above. .. The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs). .. Exonuclease III treatment was performed for 1.5 or 3 min at 15°C, and λ exonuclease treatment was performed for 1 or 2 min at 15°C.

Polymerase Chain Reaction:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After PCR, the products were purified by use of the E.Z.N.A.® Cycle-Pure Kit (Omega, USA). .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The PCR product was digested by Sma I and Bgl II , and inserted into the cloning site of pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA). .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al .

Construct:

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al . .. The digested plasmid was treated with KOD DNA polymerase (TOYOBO, Osaka, Japan) and ligated with Ligation high (TOYOBO).

Polyacrylamide Gel Electrophoresis:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

IA:

Article Title: Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion
Article Snippet: Preparation of templates All oligodeoxynucleotides were purchased from Integrated DNA Technologies, Inc (Coralville, IA, USA). .. Non-circularized linear oligonucleotides were removed by reaction with 25 U/mL exonuclease I (Takara Bio) and 1000 U/mL exonuclease III (Takara Bio) at 37 °C for 30 min.

In Situ Hybridization:

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: Paragraph title: Chromosome orientation fluorescence in situ hybridization ... The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: Paragraph title: Telomere Chromatid Orientation-Fluorescence In Situ Hybridization (CO-FISH) ... The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Plasmid Preparation:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: Then, the hybridization mixture was incubated at 95 °C for 1 min followed by immediate cooling to 40 °C and incubation for 10 min at 30 °C. .. After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: The BrdU-substituted DNA was digested with Exonuclease III (Takara). .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line
Article Snippet: The PCR product was digested by Sma I and Bgl II , and inserted into the cloning site of pGL3 Luciferase Reporter Vector (Promega, Madison, WI, USA). .. The plasmid containing the cloned fragment was digested with Kpn I and Nhe I , followed by treatment with exonuclease III (TaKaRa Bio) at 25°C for 5 to 8.5 min as described by Kerkhoff et al . .. [ ], and S1-nuclease (TaKaRa Bio) at 23°C for 15 min.

Software:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. The purified DNA was analyzed using 10% PAGE containing 7 M urea (Tris-Borate-EDTA buffer) and visualized by staining using UltraPower™ DNA/RNA Safedye (Gellex International, Tokyo, Japan) and a blue-light transilluminator.

Irradiation:

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: Chromosome slides were treated with RNase A, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml), incubated in 2 × SSC (Invitrogen) for 15 min and exposed to 365 nm UV light (Stratalinker 1800UV irradiator) for 40 min. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Negative Control:

Article Title: Amorphous nanosilica induce endocytosis-dependent ROS generation and DNA damage in human keratinocytes
Article Snippet: After 24 h, cells were treated with various concentrations of nSP70, nSP300, mSP1000, 0.2 mM H2 O2 (positive control) or PBS (negative control). .. Ten μg of DNA was converted to single stranded DNA by incubation with 180 U Exonuclease III (Takara Biotech., Japan) at 37°C for 1 h. The DNA was heated at 95°C for 5 min, rapidly chilled on ice, and digested to nucleosides by incubation with 0.6 U nuclease P1 (Takara) at 37°C for 1 h followed by treatment with 0.6 U E. coli alkaline phosphatase (Takara) for a further 1 h. The reaction mixture was centrifuged (6000 × g for 1 min) and the supernatant used for the 8-OHdG assay.

Agarose Gel Electrophoresis:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

In Vitro:

Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors
Article Snippet: After the purified eGFP -MiLV was checked by DNA sequencing, it was used for cell transfection and intramuscular injection. .. To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis. .. The EBV genome was extracted from B95-8 cells using phenol/chloroform and purified by ethanol precipitation.

Article Title: In Vitro Reconstitution of the End Replication Problem
Article Snippet: Products obtained from in vitro replication performed in the presence of [α-32 P]dATP were purified as outlined above. .. The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).

BrdU Incorporation Assay:

Article Title: Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation
Article Snippet: 5′-Bromo-2′-deoxyuridine (BrdU) was added for 18 h to allow BrdU incorporation for one cell cycle. .. The BrdU-substituted DNA was digested with 3 U/μl Exonuclease III (Takara) for 10 min at room temperature.

Concentration Assay:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. The purified DNA was analyzed using 10% PAGE containing 7 M urea (Tris-Borate-EDTA buffer) and visualized by staining using UltraPower™ DNA/RNA Safedye (Gellex International, Tokyo, Japan) and a blue-light transilluminator.

Article Title: Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system
Article Snippet: The substrate DNA was digested by exonuclease III ( TaKaRa Bio Inc., Tokyo) in 1× exonuclease III buffer at pH 8.0 (50 mM Tris–HCl, 5 mM MgCl2 , 10 mM 2-mercaptoethanol) and 37°C for 5 or 10 min. Then, 1× nuclease S1 buffer pH 4.6 (30 mM sodium acetate, 1 mM zinc acetate, 5% (v/v) glycerol) and PNA additives were added (the volume ratio of the nuclease S1 buffer to the exonuclease III buffer was 1.5:1.0). .. The substrate DNA was digested by exonuclease III ( TaKaRa Bio Inc., Tokyo) in 1× exonuclease III buffer at pH 8.0 (50 mM Tris–HCl, 5 mM MgCl2 , 10 mM 2-mercaptoethanol) and 37°C for 5 or 10 min. Then, 1× nuclease S1 buffer pH 4.6 (30 mM sodium acetate, 1 mM zinc acetate, 5% (v/v) glycerol) and PNA additives were added (the volume ratio of the nuclease S1 buffer to the exonuclease III buffer was 1.5:1.0).

Staining:

Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection
Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA). .. After enzyme inactivation at 80 °C for 15 min, the circular ssDNA was purified using a High Pure PCR Cleanup Micro Kit (Roche Diagnostics GmbH, Mannheim, Germany).

Article Title: Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells
Article Snippet: Chromosome slides were treated with RNaseA, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml) for 15 min and exposed to 365 nm UV light for 40 min. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Tcstv1 and Tcstv3 elongate telomeres of mouse ES cells
Article Snippet: Chromosome slides were treated with RNase A, fixed with 4% formaldehyde, then stained with Hoechst 33258 (0.5 mg/ml), incubated in 2 × SSC (Invitrogen) for 15 min and exposed to 365 nm UV light (Stratalinker 1800UV irradiator) for 40 min. .. The BrdU-substituted DNA was digested with Exonuclease III (Takara).

Article Title: Zscan4 promotes genomic stability during reprogramming and dramatically improves the quality of iPS cells as demonstrated by tetraploid complementation
Article Snippet: Slides were stained with 0.5 μg/ml Hoechst 33258 (Sigma), washed in 2× SSC, and then exposed to longwave radiation (e.g., 365 nm) in McIlvaine's buffer (pH 8.0) for 30 min. .. The BrdU-substituted DNA was digested with 3 U/μl Exonuclease III (Takara) for 10 min at room temperature.

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    TaKaRa exonuclease iii
    <t>Exosomes</t> secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from <t>three</t> independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.
    Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/TaKaRa
    Average 93 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2019-10
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    77
    TaKaRa escherichia coli exonuclease iii
    Exonuclease <t>III</t> digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by <t>Escherichia</t> coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.
    Escherichia Coli Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli exonuclease iii/product/TaKaRa
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    escherichia coli exonuclease iii - by Bioz Stars, 2019-10
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    Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosomes secretion prevents ATM/ATR-dependent DDR. Pre-senescent TIG-3 cells were transfected with two different sets of validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ) or to cell proliferation analysis ( b ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. Error bars indicate mean±s.d. of triplicate measurements.

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot

    Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in mouse liver. ICR mice were subjected to hydrodynamic tail vein injection with plasmid encoding firefly luciferase or small hairpin RNA (shRNA) against Alix or control ( n =3 per group). After 48 h, the mice transfected with firefly luciferase were subjected to i n vivo bioluminescent imaging for confirmation of the transfection efficiency ( a ), and then other mice were euthanized and livers were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles ( c ) or to immunofluorescence analysis of liver section ( d ). Tubulin was used as a loading control ( b ). Section of livers were subjected to immunofluorescence staining for markers of DNA damage (53BP1 (red) and 4′,6-diamidino-2-phenylindole (blue)) ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for 53BP1 staining. At least 100 cells were scored per group. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Mouse Assay, Injection, Plasmid Preparation, Luciferase, shRNA, Transfection, Imaging, Western Blot, Isolation, Immunofluorescence, Staining

    Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Exosome secretion prevents viral hijacking of cellular machinery. ( a ) Timeline of the experimental procedure. ( b – e ) Pre-senescent TIG-3 cells transfected with indicated siRNA oligos followed by infection with recombinant adenovirus encoding GFP (Ad-GFP) were subjected to western blotting using antibodies shown right ( b ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( c ), quantitative measurement of isolated adenoviral DNA from exosome using quantitative PCR ( d ), or to microscopic analysis of GFP expression ( e ). The representative data from three independent experiments are shown. ( f ) Timeline of the experimental procedure. ( g – i ) 293 cells were transfected with indicated siRNA oligos followed by infection with Ad-GFP. These cells were then subjected to western blotting using antibodies shown right ( g ), NanoSight analysis (NTA) and western blotting against canonical exosome markers for quantitative measurement of isolated exosome particles ( h ) or to titration of generated Ad-GFP ( i ). The histograms indicate the virus titre ( i ). For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Infection, Recombinant, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Titration, Generated

    Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Inhibition of exosome secretion in pre-senescent HDFs. ( a ) Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right (WCL) or to exosome isolation followed by western blotting using antibodies against canonical exosome markers shown right (exosome) and NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles. The representative data from three independent experiments are shown. Tubulin was used as a loading control. ( b – d ) Pre-senescent TIG-3 cells cultured under the conditions described in a were subjected to cell proliferation analysis ( b ), apoptosis analysis at day 4 ( c ) or to immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 4′,6-diamidino-2-phenylindole [blue]) ( d ). The representative data from three independent experiments are shown. The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). ( e , f ) Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged wild-type Alix or Rab27a protein containing a mutated siRNA cleavage site (lanes 3 and 4) or empty vector (lanes 1 and 2). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right, NanoSight analysis for quantitative measurement of isolated exosome particles, apoptosis analysis at day 4 or to immunofluorescence staining for markers of DNA damage. Tubulin was used as a loading control. The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Inhibition, Transfection, Western Blot, Isolation, Cell Culture, Immunofluorescence, Staining, Infection, Plasmid Preparation, Selection

    Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Overexpression of Dnase2a attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were infected with retrovirus encoding flag-tagged Dnase2a (lanes 4–6) or empty vector (lanes 1–3). After selection with puromycin, cells were transfected with indicated siRNA oligos and then subjected to western blotting using antibodies shown right ( a ), NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and western blotting using antibodies against canonical exosome markers shown right (exosome) ( b ), isolation of cytoplasmic fraction followed by quantitative PCR (qPCR) analysis of chromosomal DNA ( c ), immunofluorescence staining for markers of DNA damage (γ-H2AX [red], pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( d ), qPCR analysis of IFNβ gene expression ( e ), analysis of intracellular ROS levels ( e ) or to apoptosis analysis at day 4 ( e ). Tubulin was used as a loading control ( a ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( d ). At least 100 cells were scored per group ( d ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (** P

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Over Expression, Infection, Plasmid Preparation, Selection, Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Expressing

    Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

    doi: 10.1038/ncomms15287

    Figure Lengend Snippet: Reduction of ROS levels attenuated the effects of Alix or Rab27a knockdown in HDFs. Pre-senescent TIG-3 cells were transfected with validated siRNA oligos indicated at the top of the panel for two times at 2 day intervals in the presence or absence of 1 mM N -acetyl cysteine. These cells were then subjected to western blotting using antibodies shown right ( a ), analysis of intracellular ROS levels ( b ), immunofluorescence staining for markers of DNA damage (γ-H2AX (red), pST/Q (green) and 4′,6-diamidino-2-phenylindole (blue)) ( c ) or to apoptosis analysis ( d ). The histograms indicate the percentage of nuclei that contain more than 3 foci positive for both γ-H2AX and pST/Q staining ( c ). At least 100 cells were scored per group ( c ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean±s.d. of triplicate measurements. (* P

    Article Snippet: To reduce external DNA contamination, prior to DNA extraction, exosomes were treated with DNase I (Roche Inc.) and Exonuclease III (Takara Inc.), according to the manufacturers' instructions .

    Techniques: Transfection, Western Blot, Immunofluorescence, Staining

    Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p

    Journal: PLoS ONE

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

    doi: 10.1371/journal.pone.0047159

    Figure Lengend Snippet: Expression ratio and duration of GFP in different eukaryotic cells transfected with equal molar of pEGFP-N3 plasmid or eGFP -MiLV. A: Average ratio of GFP+ cells in HEK 293, NIH 3T3, CNE2 cells upon transfection with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV for 48 h; B: The duration of GFP expression in HEK 293, NIH 3T3, CNE2 cells; C: The GFP fluorescence intensity AU (arbitrary units) of HEK 293 cells after transfected with 0.1 µM DNA/cm 2 pEGFP-N3 plasmid or eGFP -MiLV. Data are representative of at least three experiments. *p

    Article Snippet: To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

    Techniques: Expressing, Transfection, Plasmid Preparation, Fluorescence

    Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p

    Journal: PLoS ONE

    Article Title: A Novel Micro-Linear Vector for In Vitro and In Vivo Gene Delivery and Its Application for EBV Positive Tumors

    doi: 10.1371/journal.pone.0047159

    Figure Lengend Snippet: Effect of DNA-induced pro-inflammatory cytokines on GFP in the blood after intravenous injection eGFP -MiLV and pEGFP-N3 plasmid. Mice received an intravenous injection of 40 µg eGFP -MiLV and pEGFP-N3 plasmid. At 2 h after injection, the levels of TNF-α, IL-6 and IL-12 in blood were measured. The results are expressed at the mean ± SD of three mice. * p

    Article Snippet: To examine the in vitro resistance of MiLV to exonuclease, the eGFP -MiLV and eGFP fragment were incubated with Exonuclease III (Takara, Japan) at 37°C for 2 to 24 h and detected by 1% agarose gel electrophoresis.

    Techniques: Injection, Plasmid Preparation, Mouse Assay

    Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.

    Journal: Scientific Reports

    Article Title: RNase H-assisted RNA-primed rolling circle amplification for targeted RNA sequence detection

    doi: 10.1038/s41598-018-26132-x

    Figure Lengend Snippet: Comparison of the effect of the position of the padlock probe. ( A ) Graphical representation of each padlock probe position on in vitro -transcribed GFP mRNA. The numbers above each probe indicate the nucleotide numbers from the 5′ end of mRNA. The letters in each padlock probe indicate the nucleotide pair of the arm end (left, 5′ end; right, 3′ end). ( B ) Denatured polyacrylamide gel analysis of ligation products using padlock probes with different hybridizing sequences on the mRNA sequence. M, 20-bp DNA size marker. The black arrow indicates a circularized padlock probe. The red arrows indicate oligo debris of DNase treatment. L, linear probe; C, circular padlock probe made by CircLigase. ( C ) Relative yield of each circularized padlock probe. The gel images were analyzed using ImageJ for comparisons with the known concentration of pre-circularized probe made by CircLigase (Circ.) as a control. The yield of each padlock probe was calculated by setting the control as 100%. Three electrophoresis experiments were conducted with three independent reaction products, and the mean and error were calculated.

    Article Snippet: After hybridization, 10 µl of a ligase mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM magnesium acetate (MgAc), 50 mM KGlu, 20, 800, or 2000 µM ATP, and each ligase (200 units of T4Dnl, 5 units of T4Rnl2, and 12.5 units of SplintR ligase, New England BioLabs) were added to the hybridization mixture and then incubated at 37 °C for 1 h to seal the padlock probe, followed by enzyme inactivation at 65 °C for 10 min. After ligation, 10 µl of a nuclease mixture containing 20 mM Tris-acetate (pH 7.5), 20 mM MgAc, 50 mM KGlu, 1 mM ATP, 10 units of Plasmid-Safe DNase (Epicentre Technologies, Madison, WI, USA), 10 units of exonuclease III (Takara Bio), and 10 units of exonuclease I (New England BioLabs) were added to the ligation mixture, which was then incubated at 37 °C for 15 min to degrade linear single-stranded DNA (ssDNA).

    Techniques: In Vitro, Ligation, Sequencing, Marker, Concentration Assay, Electrophoresis

    Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Journal: FEBS Open Bio

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes

    doi: 10.1016/j.fob.2013.08.007

    Figure Lengend Snippet: Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Article Snippet: Briefly, each reconstituted nucleosome, containing tlH2A, tlH2B, tlH3, or tlH4, was treated with 5 units of Escherichia coli exonuclease III (Takara), in 10 μl of 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2 , and 1 mM DTT.

    Techniques: Purification, Polyacrylamide Gel Electrophoresis