Image: Steps of Molecular Cloning

Description: The overall goal of molecular cloning is to take a gene of interest from one plasmid (original plasmid) and insert it into another plasmid (recombinant plasmid) and have that plasmid replicate inside a host cell (usually a bacterial cell, but can be a eukaryotic cell). First, the gene of interest is (1) amplified using PCR then (2) cut out of the plasmid using restriction enzymes that will only cut a particular sequence of nucleotides. This results in a separate linear vector and insert. The gene of interest is then (3) inserted into another vector using DNA ligase. This acts to glue the insert into the vector, resulting in a recombinant plasmid. This plasmid is then (4) transformed into a competent bacterial cell. The bacteria is then grown for a brief time in lysogeny broth and (5) spread onto an agar plate and incubated overnight. This plate contains a specific antibiotic. The recombinant plasmid contains a gene (antibiotic resistance gene) that allows the bacteria to grow on the plate. The plates are then checked (6) for colonies. Only the bacterial cells that were successfully transformed will contain the plasmid with the antibiotic resistance gene and be able to colonize. Reference: https://www.ncbi.nlm.nih.gov/books/NBK26837/
Title: Steps of Molecular Cloning
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Author: Alexpicardal97
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