Incubation:Article Title: Structural and functional insights into internal domain replacement in SpCas9 for protein engineering
Article Snippet: .. Subsequently, NEBuffer 4 and 1 unit of endonuclease V (NEB) were added to each sample, and the mixture was incubated at 37 °C for 1 h. Samples were then mixed with TBE-Urea denaturing sampling buffer (1 × TBE, 3.5 M urea, 0.05% (w/v) bromophenol blue, and 5% Ficoll) and heated at 95 °C for 5 min to inactivate the enzymes and denature the DNA. .. The samples were loaded onto a 12% TBE-Urea denaturing polyacrylamide gel and subjected to electrophoresis at 200 V for 40 min. Cleaved fractions were visualized and quantified using the ChemiDoc MP system, and data analysis was performed using GraphPad Prism.
Article Title: Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.
Article Snippet: .. Subsequently, NEBuffer 4 and 1 unit of endonuclease V (NEB) were added to each sample, and the mixture was incubated at 37 °C for 1 h. Samples were then mixed with TBE-Urea denaturing sampling buffer (1 × TBE, 3.5 M urea, 0.05% (w/v) bromophenol blue, and 5% Ficoll) and heated at 95 °C for 5 min to inactivate the enzymes and denature the DNA. .. The samples were loaded onto a 12% TBE-Urea denaturing polyacrylamide gel and subjected to electrophoresis at 200 V for 40 min. Cleaved fractions were visualized and quantified using the ChemiDoc MP system, and data analysis was performed using GraphPad Prism.
Article Title: Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity using CHANGE-seq-BE.
Article Snippet: .. A total of 3 μg of purified in vitro ABE-deaminated DNA was mixed with 4 μl of endonuclease V (10 U per μl) and 20 μl of NEB buffer 4 (10×) in a final volume of 200 μl with nuclease-free water and incubated at 37 °C for 2 h. On the other hand, 2 μg of purified in vitro CBE-deaminated DNA was treated with 6 μl of USER (1 U per μl) and 10 μl of NEB cut smart buffer (10×) in a final volume of 100 μl with nuclease-free water and incubated at 37 °C for 3 h. Next, the final reaction was treated with proteinase K (NEB) at 37 °C for 15 min, followed by 1× volume of bead purification, and quantified by Qubit HS. .. A total of 1 μg of in vitro deaminated gDNA was fragmented to the 550–750 bp using Covaris (Life Technologies) with the following settings: duty factor, 20%; peak displayed power, 50 W; cycles per burst, 200; duration, 65 s; temperature, 20 °C.
Article Title: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina
Article Snippet: After cooling, the mixtures were treated with 1 μl of RNase A (20 mg ml −1 , MilliporeSigma) and 1 μl of proteinase K (20 mg ml −1 , Viagen Biotech 501-PK) for 15 min at room temperature; then DNA products were purified using an Oligo Clean and Concentrator kit (Zymo Research, D4061). .. Purified DNA was nicked with 5 units of Endonuclease V (EndoV, NEB) for 2-3 h at 37 °C, after which the reaction was quenched by addition of TriTrack DNA-loading dye (1 × final) (Thermo Fisher, R1161) and incubation at 95 °C for 2 min. .. The cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis with urea (Urea-PAGE) in Bio-Rad MiniProtean continuous hand cast 15% acrylamide gels in Tris-borate-EDTA (TBE; Bio-Rad, 1610770) with 7 M urea and 5% crosslinker (acrylamide:bis-acrylamide ratio of 19:1; Bio-Rad,1610144).
Sampling:Article Title: Structural and functional insights into internal domain replacement in SpCas9 for protein engineering
Article Snippet: .. Subsequently, NEBuffer 4 and 1 unit of endonuclease V (NEB) were added to each sample, and the mixture was incubated at 37 °C for 1 h. Samples were then mixed with TBE-Urea denaturing sampling buffer (1 × TBE, 3.5 M urea, 0.05% (w/v) bromophenol blue, and 5% Ficoll) and heated at 95 °C for 5 min to inactivate the enzymes and denature the DNA. .. The samples were loaded onto a 12% TBE-Urea denaturing polyacrylamide gel and subjected to electrophoresis at 200 V for 40 min. Cleaved fractions were visualized and quantified using the ChemiDoc MP system, and data analysis was performed using GraphPad Prism.
Article Title: Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.
Article Snippet: .. Subsequently, NEBuffer 4 and 1 unit of endonuclease V (NEB) were added to each sample, and the mixture was incubated at 37 °C for 1 h. Samples were then mixed with TBE-Urea denaturing sampling buffer (1 × TBE, 3.5 M urea, 0.05% (w/v) bromophenol blue, and 5% Ficoll) and heated at 95 °C for 5 min to inactivate the enzymes and denature the DNA. .. The samples were loaded onto a 12% TBE-Urea denaturing polyacrylamide gel and subjected to electrophoresis at 200 V for 40 min. Cleaved fractions were visualized and quantified using the ChemiDoc MP system, and data analysis was performed using GraphPad Prism.
Purification:Article Title: Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity using CHANGE-seq-BE.
Article Snippet: .. A total of 3 μg of purified in vitro ABE-deaminated DNA was mixed with 4 μl of endonuclease V (10 U per μl) and 20 μl of NEB buffer 4 (10×) in a final volume of 200 μl with nuclease-free water and incubated at 37 °C for 2 h. On the other hand, 2 μg of purified in vitro CBE-deaminated DNA was treated with 6 μl of USER (1 U per μl) and 10 μl of NEB cut smart buffer (10×) in a final volume of 100 μl with nuclease-free water and incubated at 37 °C for 3 h. Next, the final reaction was treated with proteinase K (NEB) at 37 °C for 15 min, followed by 1× volume of bead purification, and quantified by Qubit HS. .. A total of 1 μg of in vitro deaminated gDNA was fragmented to the 550–750 bp using Covaris (Life Technologies) with the following settings: duty factor, 20%; peak displayed power, 50 W; cycles per burst, 200; duration, 65 s; temperature, 20 °C.
Article Title: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina
Article Snippet: After cooling, the mixtures were treated with 1 μl of RNase A (20 mg ml −1 , MilliporeSigma) and 1 μl of proteinase K (20 mg ml −1 , Viagen Biotech 501-PK) for 15 min at room temperature; then DNA products were purified using an Oligo Clean and Concentrator kit (Zymo Research, D4061). .. Purified DNA was nicked with 5 units of Endonuclease V (EndoV, NEB) for 2-3 h at 37 °C, after which the reaction was quenched by addition of TriTrack DNA-loading dye (1 × final) (Thermo Fisher, R1161) and incubation at 95 °C for 2 min. .. The cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis with urea (Urea-PAGE) in Bio-Rad MiniProtean continuous hand cast 15% acrylamide gels in Tris-borate-EDTA (TBE; Bio-Rad, 1610770) with 7 M urea and 5% crosslinker (acrylamide:bis-acrylamide ratio of 19:1; Bio-Rad,1610144).
In Vitro:Article Title: Sensitive and unbiased genome-wide profiling of base-editor-induced off-target activity using CHANGE-seq-BE.
Article Snippet: .. A total of 3 μg of purified in vitro ABE-deaminated DNA was mixed with 4 μl of endonuclease V (10 U per μl) and 20 μl of NEB buffer 4 (10×) in a final volume of 200 μl with nuclease-free water and incubated at 37 °C for 2 h. On the other hand, 2 μg of purified in vitro CBE-deaminated DNA was treated with 6 μl of USER (1 U per μl) and 10 μl of NEB cut smart buffer (10×) in a final volume of 100 μl with nuclease-free water and incubated at 37 °C for 3 h. Next, the final reaction was treated with proteinase K (NEB) at 37 °C for 15 min, followed by 1× volume of bead purification, and quantified by Qubit HS. .. A total of 1 μg of in vitro deaminated gDNA was fragmented to the 550–750 bp using Covaris (Life Technologies) with the following settings: duty factor, 20%; peak displayed power, 50 W; cycles per burst, 200; duration, 65 s; temperature, 20 °C.
Amplification:Article Title: Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice
Article Snippet: Deaminated DNA products were treated with proteinase K (NEB), followed by Endonuclease V (NEB) treatment. .. The Endonuclease V-treated products were end repaired with Klenow fragment exo-(NEB), A-tailed (NEB), ligated with a hairpin adapter (NEB), treated with USER enzyme (NEB) and amplified by PCR using KAPA HiFi HotStart Uracil+ Ready Mix (Roche). .. Completed libraries were quantified by qPCR using KAPA Library Quantification kit (Complete kit; Universal) (Roche) and sequenced with 151-bp paired-end reads on an Illumina NextSeq 2000 instrument.
Polymerase Chain Reaction:Article Title: Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice
Article Snippet: Deaminated DNA products were treated with proteinase K (NEB), followed by Endonuclease V (NEB) treatment. .. The Endonuclease V-treated products were end repaired with Klenow fragment exo-(NEB), A-tailed (NEB), ligated with a hairpin adapter (NEB), treated with USER enzyme (NEB) and amplified by PCR using KAPA HiFi HotStart Uracil+ Ready Mix (Roche). .. Completed libraries were quantified by qPCR using KAPA Library Quantification kit (Complete kit; Universal) (Roche) and sequenced with 151-bp paired-end reads on an Illumina NextSeq 2000 instrument.
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