Polymerase Chain Reaction:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Sterility:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Article Title: Single Genomic Loci Labeling and Manipulation Using SIMBA System.
Article Snippet: .. Materials Addgene plasmids (Table 1) 1× ampicillin LB agar plates (see recipe) Stbl3 competent cells (Invitrogen, C737303) S.O.C. medium (Invitrogen, 15544034) 1× ampicillin LB culture medium (see recipe) 50% (w/v) glycerol, sterile (filter-sterilize with 0.22-μm filter) GenElute HP Plasmid DNA Miniprep Kit (Sigma, NA0150-1KT; for plasmid extraction from bacterial culture) Molecular biology–grade water (Fisher, BP2819-1) Appropriate primers for cassette cloning(Table 2) Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491L) 10× rCutSmart Buffer (New England Biolabs, B6004; for restriction enzyme reactions) Restriction enzymes (New England Biolabs): EcoRI (EcoRI-HF, R3101S), NotI (NotI-HF, R3189S), BamHI (BamHI-HF, R3136S), and XhoI(R0146S) for Gibson Assembly subcloning and plasmid digestion fingerprint identification BbsI (R3539S), for Golden Gate Assembly Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) Gibson Assembly Master Mix (New England Biolabs, E2611L; for DNA recombination) Chen et al. 3 of 23 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.947, W iley O nline L ibrary on [29/12/2023]. ..
Cloning:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Article Title: Single Genomic Loci Labeling and Manipulation Using SIMBA System.
Article Snippet: .. Materials Addgene plasmids (Table 1) 1× ampicillin LB agar plates (see recipe) Stbl3 competent cells (Invitrogen, C737303) S.O.C. medium (Invitrogen, 15544034) 1× ampicillin LB culture medium (see recipe) 50% (w/v) glycerol, sterile (filter-sterilize with 0.22-μm filter) GenElute HP Plasmid DNA Miniprep Kit (Sigma, NA0150-1KT; for plasmid extraction from bacterial culture) Molecular biology–grade water (Fisher, BP2819-1) Appropriate primers for cassette cloning(Table 2) Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491L) 10× rCutSmart Buffer (New England Biolabs, B6004; for restriction enzyme reactions) Restriction enzymes (New England Biolabs): EcoRI (EcoRI-HF, R3101S), NotI (NotI-HF, R3189S), BamHI (BamHI-HF, R3136S), and XhoI(R0146S) for Gibson Assembly subcloning and plasmid digestion fingerprint identification BbsI (R3539S), for Golden Gate Assembly Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) Gibson Assembly Master Mix (New England Biolabs, E2611L; for DNA recombination) Chen et al. 3 of 23 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.947, W iley O nline L ibrary on [29/12/2023]. ..
Sequencing:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Plasmid Preparation:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Article Title: Single Genomic Loci Labeling and Manipulation Using SIMBA System.
Article Snippet: .. Materials Addgene plasmids (Table 1) 1× ampicillin LB agar plates (see recipe) Stbl3 competent cells (Invitrogen, C737303) S.O.C. medium (Invitrogen, 15544034) 1× ampicillin LB culture medium (see recipe) 50% (w/v) glycerol, sterile (filter-sterilize with 0.22-μm filter) GenElute HP Plasmid DNA Miniprep Kit (Sigma, NA0150-1KT; for plasmid extraction from bacterial culture) Molecular biology–grade water (Fisher, BP2819-1) Appropriate primers for cassette cloning(Table 2) Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491L) 10× rCutSmart Buffer (New England Biolabs, B6004; for restriction enzyme reactions) Restriction enzymes (New England Biolabs): EcoRI (EcoRI-HF, R3101S), NotI (NotI-HF, R3189S), BamHI (BamHI-HF, R3136S), and XhoI(R0146S) for Gibson Assembly subcloning and plasmid digestion fingerprint identification BbsI (R3539S), for Golden Gate Assembly Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) Gibson Assembly Master Mix (New England Biolabs, E2611L; for DNA recombination) Chen et al. 3 of 23 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.947, W iley O nline L ibrary on [29/12/2023]. ..
Extraction:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Article Title: Single Genomic Loci Labeling and Manipulation Using SIMBA System.
Article Snippet: .. Materials Addgene plasmids (Table 1) 1× ampicillin LB agar plates (see recipe) Stbl3 competent cells (Invitrogen, C737303) S.O.C. medium (Invitrogen, 15544034) 1× ampicillin LB culture medium (see recipe) 50% (w/v) glycerol, sterile (filter-sterilize with 0.22-μm filter) GenElute HP Plasmid DNA Miniprep Kit (Sigma, NA0150-1KT; for plasmid extraction from bacterial culture) Molecular biology–grade water (Fisher, BP2819-1) Appropriate primers for cassette cloning(Table 2) Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491L) 10× rCutSmart Buffer (New England Biolabs, B6004; for restriction enzyme reactions) Restriction enzymes (New England Biolabs): EcoRI (EcoRI-HF, R3101S), NotI (NotI-HF, R3189S), BamHI (BamHI-HF, R3136S), and XhoI(R0146S) for Gibson Assembly subcloning and plasmid digestion fingerprint identification BbsI (R3539S), for Golden Gate Assembly Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) Gibson Assembly Master Mix (New England Biolabs, E2611L; for DNA recombination) Chen et al. 3 of 23 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.947, W iley O nline L ibrary on [29/12/2023]. ..
Bacteria:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Spectrophotometry:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Concentration Assay:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Software:Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. Materials pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders Prepare the Cas9 plasmid list-behavior=simple prefix-word= mark-type=none max-label-size=1 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock. ..
Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells
Article Snippet: .. pSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459 v2.0; Addgene, cat. No. 62988) LB agar plates containing 100 μg/mL ampicillin LB liquid medium containing 100 μg/mL ampicillin LB agar plates containing 50 μg/mL kanamycin LB liquid medium containing 50 μg/mL kanamycin PCR-grade sterile deionized water Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. K2800-20) One Shot Top10 Chemically Competent E. coli cells (Thermo Fisher Scientific, cat. no. C404010) M13 Forward (5'-GTTTTCCCAGTCACGACG-3') and M13 Reverse (5'-AACAGCTATGACCATG-3') universal sequencing primers (included with Zero Blunt TOPO PCR Cloning Kit) Plasmid Extraction Mini Kit and Midi Kit (Qiagen) Sterile pipet tips for picking colonies from agar plates 37°C bacterial incubator-shaker 45°C incubator for heat-shocking bacteria Nanodrop micro spectrophotometer, or another device for measuring DNA concentration DNA Sequence analysis software (e.g., NCBI BLAST, UCSC Genome Browser BLAT, DNASTAR LaserGene Suite) 10-mL bacterial culture tubes Standard 1.5 mL Eppendorf tubes Access to Sanger sequencing facility L-shaped bacterial spreaders .. 1 From Addgene, order the selected Cas9 plasmid, which will arrive as a bacterial stock.
Subcloning:Article Title: Single Genomic Loci Labeling and Manipulation Using SIMBA System.
Article Snippet: .. Materials Addgene plasmids (Table 1) 1× ampicillin LB agar plates (see recipe) Stbl3 competent cells (Invitrogen, C737303) S.O.C. medium (Invitrogen, 15544034) 1× ampicillin LB culture medium (see recipe) 50% (w/v) glycerol, sterile (filter-sterilize with 0.22-μm filter) GenElute HP Plasmid DNA Miniprep Kit (Sigma, NA0150-1KT; for plasmid extraction from bacterial culture) Molecular biology–grade water (Fisher, BP2819-1) Appropriate primers for cassette cloning(Table 2) Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491L) 10× rCutSmart Buffer (New England Biolabs, B6004; for restriction enzyme reactions) Restriction enzymes (New England Biolabs): EcoRI (EcoRI-HF, R3101S), NotI (NotI-HF, R3189S), BamHI (BamHI-HF, R3136S), and XhoI(R0146S) for Gibson Assembly subcloning and plasmid digestion fingerprint identification BbsI (R3539S), for Golden Gate Assembly Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) Gibson Assembly Master Mix (New England Biolabs, E2611L; for DNA recombination) Chen et al. 3 of 23 Current Protocols nloaded from https://currentprotocols.onlinelibrary.w iley.com /doi/10.1002/cpz1.947, W iley O nline L ibrary on [29/12/2023]. ..
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