Standard molecular biology techniques were used to generate all constructs analyzed in this work. Constructs were built by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Cat. # E2621). Tables listing the PCR template and a pair of oligos used to amplify each DNA fragment used to build plasmids generated in this work can be found in the “Supplementary Methods” section in the Supplementary Information file. After assembly, plasmids were transformed into NEB 5-alpha Electrocompetent Competent E. coli (New England Biolabs, Cat. # C2989). Correct clones were subsequently confirmed by restriction analysis and Sanger sequencing. The final DNA sequence information for the constructs is available on NCBI; accession numbers are provided for each construct in the Supplementary Information file.
Neb 5 alpha electrocompetent competent e coli
Manufactured by New England Biolabs
NEB 5-alpha Electrocompetent Competent E. coli is a laboratory strain of Escherichia coli bacteria that has been made electrocompetent, allowing for efficient transformation of DNA through electroporation. The strain is suitable for a variety of molecular biology applications that require the introduction of recombinant DNA.
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Lab products found in correlation
4 protocols using neb 5 alpha electrocompetent competent e coli
1
DNA Construct Generation by Gibson Assembly
2
Culex Mosquito Genetic Manipulation
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Standard molecular biological techniques were applied to generate all constructs analyzed in this work. The genomic DNA of ~30 adult wild-type Culex. quinquefasciatus was extracted using DNeasy Blood & Tissue Kit (Qiagen, # 69504). For Culex promoters used in this study, their 5′ regulatory and 3′ UTR sequences were amplified with Q5 Hot Start High-Fidelity 2X Master Mix (New England, USA, # M0494S), and cloned on each respective side of either a Cas9 gene or a gRNA preceded by a double-BbsI (or in one case BsmbI) restriction site linker for later insertion of different gRNAs. Plasmids were built by Gibson Assembly using NEBuilder Hifi DNA Assembly Master Mix (New England Biolabs, # E2621). After assembling, the plasmids were transformed into NEB 5-alpha Electrocompetent Competent E. coli (New England Biolabs, # C2989). Correct clones were subsequently confirmed by restriction digestion and Sanger sequencing. All primers used to build plasmids generated in this work are listed in Supplementary Table 2 .
3
Generation of a Cq-U6-1-w6-gRNA Plasmid
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The white-gRNA6 fragment was synthesized by annealing oligos, and inserted into the double-BbsI restriction site linker of Cq-U6-1_2XBbsI-gRNA plasmid (Addgene#169238) to build a Cq-U6-1-w6-gRNA plasmid. The gRNA scaffold is modified by the insertion of an extra 5bp loop structure to boost transgenesis as mentioned in our previous studies26 (link). The Cq-U6-1-w6-gRNA fragment was later amplified and cloned into the white (CPIJ005542) gene flanked by the homology arms abutting the w6-gRNA cutting site. The construct was built by Gibson Assembly using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, #E2621). After assembling, the plasmid was transformed into NEB 5-alpha Electrocompetent Competent E. coli (New England Biolabs, # C2989) and correct clones were subsequently confirmed by restriction digestion and Sanger sequencing. All primers used to build plasmids generated in this work are listed in Supplementary Table 1 .
4
Culex quinquefasciatus Genomic DNA Extraction and Plasmid Construction
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Standard molecular biological techniques were applied to generate all constructs analyzed in this work. The genomic DNA of ~30 adult wild-type Culex. quinquefasciatus was extracted using DNeasy Blood & Tissue Kit (Qiagen, # 69504). For Culex promoters used in this study, their 5' regulatory and 3' UTR sequences were amplified with Q5 Hot Start High-Fidelity 2X Master Mix (New England, USA, # M0494S), and cloned on each respective side of either a Cas9 gene or a gRNA preceded by a double-BbsI (or in one case BsmbI ) restriction site linker for later insertion of different gRNAs. Plasmids were built by Gibson Assembly using NEBuilder Hifi DNA Assembly Master Mix (New England Biolabs, # E2621). After assembling, the plasmids were transformed into NEB 5-alpha Electrocompetent Competent E. coli (New England Biolabs, # C2989). Correct clones were subsequently confirmed by restriction digestion and Sanger sequencing. All primers used to build plasmids generated in this work are listed in Supplementary Methods .
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