Gel Extraction:Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) MgCl2 (25 mM; ThermoScientific, cat. no. R0971) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAprep spin miniprep kit (Qiagen, cat. no. 27106) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) SYBR Safe DNA stain (10,000×; Life Technologies, cat. no. 533102) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest BbsI (BpiI) (Fermentas/ThermoScientific, cat. no. FD1014) Fermentas Tango Buffer (Fermentas/ThermoScientific, cat. no. BY5) DL-dithiothreitol (DTT; Fermentas/ThermoScientific, cat. no. R0862) T7 DNA ligase (Enzymatics, cat. no. L602L) Critical: Do not substitute the more commonly used T4 ligase. ..
Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Staining:Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) MgCl2 (25 mM; ThermoScientific, cat. no. R0971) QIAquick gel extraction kit (Qiagen, cat. no. 28704) QIAprep spin miniprep kit (Qiagen, cat. no. 27106) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) SYBR Safe DNA stain (10,000×; Life Technologies, cat. no. 533102) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest BbsI (BpiI) (Fermentas/ThermoScientific, cat. no. FD1014) Fermentas Tango Buffer (Fermentas/ThermoScientific, cat. no. BY5) DL-dithiothreitol (DTT; Fermentas/ThermoScientific, cat. no. R0862) T7 DNA ligase (Enzymatics, cat. no. L602L) Critical: Do not substitute the more commonly used T4 ligase. ..
Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Injection:Article Title: Modular gene tagging in C. elegans
Article Snippet: .. EG6699 ( ttTi5605 I; unc-119(ed3) III ) was injected with a mix of 10ng/μL pCFJ1532, 10ng/μL pCFJ104, 10ng/μL pGH8, 10ng/μL pMA122, 2.5ng/μL pCFJ90, 47.5ng/μL Invitrogen 1 Kb Plus DNA Ladder (#10787018), and 10ng/μL pMR547 (Extended Data Table 4). ..
Article Title: Modular gene tagging in C. elegans
Article Snippet: .. The EG8078 ( oxTi185[universal-Mos] I; unc-119(ed3) III) strain was injected with a mix of 10ng/μL pCFJ1532 Mosase, 10ng/μL pMR564 (FRT::Rol::FRT Cb- unv-119+) , 10ng/μL pCFJ104 P myo-3::mChr , 10ng/μL pGH8 P rab-3::mChr , 2.5ng/μL pCFJ90 P myo-2::mChr , 10ng/μL pMA122 peel-1 negative selection, and 47.5ng/μL Invitrogen 1 Kb Plus DNA Ladder (#10787018) (Extended Data Table 4). ..
Mutagenesis:Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Nucleic Acid Electrophoresis:Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Sterility:Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Transferring:Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Polymerase Chain Reaction:Article Title: Functional genomics using CRISPR-Cas systems, compositions, methods, knock out libraries and applications thereof
Article Snippet: Applicants typically transfected 2×105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat no. 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well: BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom, BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. 23| Applicants spun down the new tube at 200×g for 5 m. Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa.
Article Title: Optimized CRISPR-CAS double nickase systems, methods and compositions for sequence manipulation
Article Snippet: Purified PCR products may be stored at 4° C. or −20° C. (B) Cloning sgRNA into Cas9-Containing Bicistronic Expression Vector ⋅ Timing 3 d (i) Prepare the sgRNA oligo inserts. .. Herculase II reaction buffer (5×; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L) QIAquick gel extraction kit (Qiagen, cat. no. 28704) Taq Buffer (10×; Genscript, cat. no. B0005) SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025) UltraPure TBE buffer (10×; Life Technologies, cat. no. 15581-028) SeaKem LE agarose (Lonza, cat. no. 50004) 4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5×; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000×; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018) TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504) Equipment Filtered sterile pipette tips (Corning) Standard 1.5 ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC) Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250) Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097) Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no, 352070) Round-bottom Tube with cell strainer cap, 5 ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm×15 mm; BD Biosciences, cat. no. 351007) Tissue culture plate (24 well; BD Falcon, cat. no. 353047) Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075) Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786). .. Desktop microcentrifuges 5424, 5804 (Eppendorf) Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164-5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401) Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001) Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273) Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600) Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ, or open-source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific) Reagent Setup Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1× working solution for casting agarose gels and for use as a buffer for gel electrophoresis.
Selection:Article Title: Modular gene tagging in C. elegans
Article Snippet: .. The EG8078 ( oxTi185[universal-Mos] I; unc-119(ed3) III) strain was injected with a mix of 10ng/μL pCFJ1532 Mosase, 10ng/μL pMR564 (FRT::Rol::FRT Cb- unv-119+) , 10ng/μL pCFJ104 P myo-3::mChr , 10ng/μL pGH8 P rab-3::mChr , 2.5ng/μL pCFJ90 P myo-2::mChr , 10ng/μL pMA122 peel-1 negative selection, and 47.5ng/μL Invitrogen 1 Kb Plus DNA Ladder (#10787018) (Extended Data Table 4). ..
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