exonuclease 1 exo1  (New England Biolabs)


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    Structured Review

    New England Biolabs exonuclease 1 exo1
    Exonuclease 1 Exo1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease 1 exo1/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease 1 exo1 - by Bioz Stars, 2020-04
    93/100 stars

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    Functional Assay:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: Paragraph title: Validation, segregation analysis, and evaluating potential functional effects of mutations ... PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed.

    Magnetic Beads:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed. .. PCR extension products were purified using Agencourt CleanSeq Magnetic Beads and a SPRI plate, according to the manufacturer’s protocol (Beckman Coulter, California).

    Mutagenesis:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: Validation, segregation analysis, and evaluating potential functional effects of mutations Direct Sanger sequencing was used to confirm the detected protein-changing mutations in probands and to evaluate the segregation of the mutation in families. .. PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed.

    Size-exclusion Chromatography:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: .. PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed. .. Sequencing reactions were prepared with the respective forward primer at 5 μM and purified PCR product at 10 ng/100 bp (i.e. , 30 ng for 300 bp product) combined with BigDye Terminator v3.1 (Life Technologies) and 5× Sequencing Buffer (Life Technologies), and made up to 20 μl with water.

    Purification:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed. .. Sequencing reactions were prepared with the respective forward primer at 5 μM and purified PCR product at 10 ng/100 bp (i.e. , 30 ng for 300 bp product) combined with BigDye Terminator v3.1 (Life Technologies) and 5× Sequencing Buffer (Life Technologies), and made up to 20 μl with water.

    Polymerase Chain Reaction:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: .. PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed. .. Sequencing reactions were prepared with the respective forward primer at 5 μM and purified PCR product at 10 ng/100 bp (i.e. , 30 ng for 300 bp product) combined with BigDye Terminator v3.1 (Life Technologies) and 5× Sequencing Buffer (Life Technologies), and made up to 20 μl with water.

    Sequencing:

    Article Title: High-Throughput Genetic Screening of 51 Pediatric Cataract Genes Identifies Causative Mutations in Inherited Pediatric Cataract in South Eastern Australia
    Article Snippet: .. PCR was performed on a Palm Cycler (Corbett Life science, Qiagen) with one cycle at 95° for 5 min, followed by 30 or 35 cycles (Table S2 in File S1 ) at 95° for 30 sec, 57−65° (annealing temperature, Table S2 in File S1 ) for 30 sec, and 72° for 30 sec, and a final extension step at 72° for 5 min. To clean the PCR products for sequencing, 5 μl of PCR product, 2 μl shrimp alkaline phosphatase (SAP; 1 unit/μl) and 0.5 μl (20 units/μl) of exonuclease 1 (Exo1) (New England Biolabs, Massachusetts) were mixed. .. Sequencing reactions were prepared with the respective forward primer at 5 μM and purified PCR product at 10 ng/100 bp (i.e. , 30 ng for 300 bp product) combined with BigDye Terminator v3.1 (Life Technologies) and 5× Sequencing Buffer (Life Technologies), and made up to 20 μl with water.

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    New England Biolabs λ exonuclease
    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with <t>λ</t> exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 27 article reviews
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    99
    New England Biolabs t7 exonuclease
    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with <t>T7</t> exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    T7 Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease 1
    Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min <t>Exonuclease</t> 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.
    Exonuclease 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Incubation, Marker

    Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Journal: Analytical biochemistry

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    doi: 10.1016/j.ab.2008.06.037

    Figure Lengend Snippet: Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Article Snippet: A mixture of supercoiled (3.7 µg) and linear (3.3 µg) DNA was incubated with λ exonuclease (1 µL, 5 units/µL) and RecJf (3 µL, 30 units/µL) in a total volume of 100 µL containing 1X λ exonuclease buffer (New England Biolabs) at 37 °C for 16 h. The nicked-circular DNA preparation (14 µg) containing traces of linear DNA was then incubated again with λ exonuclease (0.5 µL) and RecJf (3 µL) in a total volume of 100 µL containing 1X λ exonuclease buffer at 37 °C for 16 h. Next, the λ exonuclease was heat-inactivated (65 °C, 10 min), and the reaction mixture was extracted with phenol and chloroform:amyl alcohol (24:1, v/v).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Marker

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    doi: 10.1074/jbc.M109.025825

    Figure Lengend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Article Snippet: G-strand overhang was also measured after incubation of genomic DNA with T7 exonuclease (1 unit/μg DNA) (New England Biolabs) in NE-Buffer 4 at 25 °C for 60 s. T7 exonuclease acts in the 5′–3′ direction to remove 5′ nucleotides from duplex DNA.

    Techniques: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression

    Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

    Journal: Communications Biology

    Article Title: Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

    doi: 10.1038/s42003-020-0768-9

    Figure Lengend Snippet: Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

    Article Snippet: In vitro binding of the repair templates to the Cas9-VirD2 and VirD2-Cas9 fusion proteins For covalent binding of the Cas9-VirD2 and VirD2-Cas9 fusion proteins to the repair templates, the repair templates (1 µM of ssDNA or 300 ng of dsDNA) were incubated with 1 µg of either Cas9, Cas9-VirD, or VirD2-Cas9 at 37 °C for 30 min. For complete binding 1 µM of ssDNA or were incubated with 1 µg of Cas9-VirD at 37 °C in MgCl2 buffer for 5 min and 1 ul of Exonuclease 1 (E. coli , NEB) was added following manufacturer’s protocol and the reaction was incubated for 25 more minutes.

    Techniques: Expressing, Purification, SDS Page, Activity Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Binding Assay, Sequencing