The reporter gene was constructed by stitching PCR products using GeneArt Seamless Cloning Technology (Life Technologies), and the product cloned into the pEGFP-N1 vector (Clontech). PCR reactions used to create wtBRAF and mutBRAF reporters were constructed using genomic DNA of parental or resistant C3 cell lines as template, respectively. Mutagenesis was performed using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). Cells were transfected using Nucleofector Technology (Lonza). Primers used in this study are provided in Table 1 .
Geneart seamless cloning technology
Manufactured by Thermo Fisher Scientific
GeneArt Seamless Cloning Technology is a tool for the efficient and precise assembly of multiple DNA fragments into a single vector. It enables the rapid construction of complex genetic constructs by facilitating the seamless joining of DNA segments without the need for restriction enzyme digestion or ligation.
Automatically generated - may contain errors
2 protocols using geneart seamless cloning technology
1
Constructing Reporter Genes via Seamless Cloning
2
Constructing Reporter Genes via Seamless Cloning
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The reporter gene was constructed by stitching PCR products using GeneArt Seamless Cloning Technology (Life Technologies), and the product cloned into the pEGFP-N1 vector (Clontech). PCR reactions used to create wtBRAF and mutBRAF reporters were constructed using genomic DNA of parental or resistant C3 cell lines as template, respectively. Mutagenesis was performed using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). Cells were transfected using Nucleofector Technology (Lonza). Primers used in this study are provided in Table 1 .
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