please refer to me the protocol of insertion of PXR gene in existing DNA which has to be transfected in HEK293 WT cell line
Fredrick Odoyo Ondiek
Isolate the PXR Gene: Obtain the PXR gene by isolating it from an existing organism through polymerase chain reaction (PCR). Prepare the Vector: need a DNA vector, such as a plasmid, to carry the PXR gene. You may need to cut the plasmid with a restriction enzyme to create a "cut" site for the PXR gene. Ligate the Gene and Vector: The PXR gene and the vector are joined together using DNA ligase, creating a recombinant plasmid. This plasmid now contains the PXR gene. Transformation: The recombinant plasmid is introduced into the host organism through transformation. For bacteria, involves heat shocking the cells to make them more permeable to DNA. For other organisms, different methods like electroporation or viral vectors may be used. Selection and Screening: Not all host cells will take up the recombinant plasmid hence a selection marker (like an antibiotic resistance gene) often includes the plasmid. Only cells that successfully incorporate the plasmid will survive when exposed to the selection agent. This allows you to screen for and identify the host cells that now contain the PXR gene. Culturing and Expression: The selected host cells are cultured to allow the PXR gene to be expressed. This means the host organism will now produce the protein encoded by the PXR gene. Verification: To confirm that the PXR gene has been successfully integrated into the host DNA, apply PCR, DNA sequencing.
Priyansha Singh, inserting a specific gene, such as the PXR gene, into existing DNA involves recombinant DNA technology. The specific method used can vary depending on the application, the host organism, and the research goals.
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Khalid Suhail A. Al-Azzawi
I agree with Fredrick Odoyo Ondiek this is a typically method to insert PXR inside the DNA of cell(s).
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Guohua Zhong
You can try FastCloning, a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.
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