Clone Assay:
Cloning:Article Title: Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery
Article Snippet: .. A cell line derivative of DH5α (NEB 5-alpha Competent E. coli , New England Biolabs, Ipswich, MA) was used for cloning the vector constructs and BL21(DE3) (NEB T7 Competent E. coli , New England Biolabs, Ipswich, MA) for protein production; sequences were confirmed through either Sanger sequencing (Eurofins Genomics, Louisville, KY) or Nanopore sequencing (Plasmidsaurus, South San Francisco, CA) of plasmid DNA. .. The plasmid sequences are provided by Addgene as well as the Supporting Information and the gBlocks are detailed in the Supporting Information (Supplementary Tables S1-2).
Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Construct:Article Title: Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery
Article Snippet: .. A cell line derivative of DH5α (NEB 5-alpha Competent E. coli , New England Biolabs, Ipswich, MA) was used for cloning the vector constructs and BL21(DE3) (NEB T7 Competent E. coli , New England Biolabs, Ipswich, MA) for protein production; sequences were confirmed through either Sanger sequencing (Eurofins Genomics, Louisville, KY) or Nanopore sequencing (Plasmidsaurus, South San Francisco, CA) of plasmid DNA. .. The plasmid sequences are provided by Addgene as well as the Supporting Information and the gBlocks are detailed in the Supporting Information (Supplementary Tables S1-2).
Article Title: Metabolic engineering enables Escherichia coli to grow on 1,3-propanediol
Article Snippet: .. Newly constructed plasmids were propagated in Dh5α (NEB C2987H) overnight and extracted in accordance with the protocol provided in the QIAprep Spin Miniprep Kit (Qiagen). .. 50 ng of the extracted genetic materials were analyzed using the nanopore sequencing workflow provided by Oxford Nanopore Technologies with Flongle Flow Cell (FLG-114) and Rapid barcoding kit (SQK-RBK114).
DNA Sequencing:Article Title: Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery
Article Snippet: .. A cell line derivative of DH5α (NEB 5-alpha Competent E. coli , New England Biolabs, Ipswich, MA) was used for cloning the vector constructs and BL21(DE3) (NEB T7 Competent E. coli , New England Biolabs, Ipswich, MA) for protein production; sequences were confirmed through either Sanger sequencing (Eurofins Genomics, Louisville, KY) or Nanopore sequencing (Plasmidsaurus, South San Francisco, CA) of plasmid DNA. .. The plasmid sequences are provided by Addgene as well as the Supporting Information and the gBlocks are detailed in the Supporting Information (Supplementary Tables S1-2).
Nanopore Sequencing:Article Title: Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery
Article Snippet: .. A cell line derivative of DH5α (NEB 5-alpha Competent E. coli , New England Biolabs, Ipswich, MA) was used for cloning the vector constructs and BL21(DE3) (NEB T7 Competent E. coli , New England Biolabs, Ipswich, MA) for protein production; sequences were confirmed through either Sanger sequencing (Eurofins Genomics, Louisville, KY) or Nanopore sequencing (Plasmidsaurus, South San Francisco, CA) of plasmid DNA. .. The plasmid sequences are provided by Addgene as well as the Supporting Information and the gBlocks are detailed in the Supporting Information (Supplementary Tables S1-2).
Plasmid Preparation:Article Title: Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery
Article Snippet: .. A cell line derivative of DH5α (NEB 5-alpha Competent E. coli , New England Biolabs, Ipswich, MA) was used for cloning the vector constructs and BL21(DE3) (NEB T7 Competent E. coli , New England Biolabs, Ipswich, MA) for protein production; sequences were confirmed through either Sanger sequencing (Eurofins Genomics, Louisville, KY) or Nanopore sequencing (Plasmidsaurus, South San Francisco, CA) of plasmid DNA. .. The plasmid sequences are provided by Addgene as well as the Supporting Information and the gBlocks are detailed in the Supporting Information (Supplementary Tables S1-2).
Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Amplification:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Polymerase Chain Reaction:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Incubation:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Bacteria:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Cell Culture:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Touchdown PCR:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
Mutagenesis:Article Title: Defective transcription of AAGAG satellite DNA causes sex-ratio meiotic drive in Drosophila.
Article Snippet: .. Briefly, the UFO12514 vector was amplified by standard PCR (95 ̊C 2 min; 30 cycles of 95 ̊C 20s, 55 ̊C 20s, 72 ̊C 30s/kb using Herculase II Fusion DNA Polymerase; 72 ̊C 3 min and 10 ̊C keep); 0.5μL vector (at least 50ng), 0.5μL insert (at least a few fold excess of insert), 3μL H2O, and 1μL In-Fusion Snap Assembly Master Mix (Takara Bio 638947) were mixed and incubated for 15 min at 50 ̊C followed by 4 ̊C keep; 50μL DH5α (NEB C2987) and 5μL In-Fusion reaction mixture was mixed and incubated for 15 min on ice, and the bacteria was heat shocked for 45 sec at 42 ̊C, on ice for 1 min, recovered in 200μL SOC (briefly if ampicillin resistant, 1h at 37 ̊C if non-ampicillin resistant), and cultured on a LB plate with 100μg/mL carbenicillin or other antibiotics overnight at 37 ̊C; Each colony is dissolved in 10μL H2O, and 1μL was used for colony PCR (mixed with19μL DreamTaq PCR mixture used for genotyping followed by touchdown PCR that amplify the insert) and 9μL was added in 200μL LB (stored at 4 ̊C during colony PCR, and cultured in 3mL LB overnight at 37 ̊C); 500μL of cultured E. coli was mixed with 500μL of 50% glycerol and stored at −80 ̊C, and the rest of cultured E. coli was used to collect plasmids using Miniprep (QIAGEN 27104; culture in 2mL LB overnight; centrifuge for 3 min; dissolve in 250μL RNase solution; add 250μL SDS solution; gently invert 5 times; incubate at RT for 3min; add 350μL neutralizing solution; gently invert 5 times; centrifuge for 10min; transfer 700μL supernatant to the spin column; add 700μL salt wash solution; centrifuge for 30sec; add 700μL ethanol wash solution; centrifuged for 30sec; centrifuge for another 1min; elute in 50μL water); The entire plasmid was sequenced by Plasmidsaurus Inc; Small cloning errors were corrected by QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent 210518). .. HP2-L fragments were amplified from testis cDNA library: Briefly, 1μL of cDNA and 5μL of 5μM forward and reverse primer mix for each fragment were added in PCR mixture (Herculase II Fusion DNA Polymerase (Agilent 600675; 1μL), 250μM dNTP (1.25μL of 10mM dNTP), 5x Buffer (10μL) in H2O (31.75μL)).
other:Article Title: Effects of CTCF on the regulatory landscape of the mouse Sox2 locus
Article Snippet: The plasmids were digested with SpeI-HF (NEB, R3133S) and NheI-HF (NEB, R3131S) and cloned in between Sleeping Beauty arms of a target vector.
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