To place your gene in the proper orientation downstream of the promoter, you can add an EcoRI site just 5’ of the start of the gene and a HindIII site just 3’ of the end of the gene. It has a promoter (blue arrow) followed by the restriction sites EcoRI, XhoI, and HindIII. Having multiple sites allows you to easily orient your gene insert with respect to the promoter.įor example, let’s say your plasmid backbone looks like the one found on the left side of the image below. Ideally, the backbone will contain a variety of restriction enzyme cut sites (restriction sites) downstream of the promoter as part of a multiple cloning site (MCS). For instance, if you were cloning a gene into an expression vector, you would want the start of the gene to end up just downstream of the promoter found in the backbone. Ligase is used to make bonds between the insert and backbone covalent.īefore beginning the restriction digest and ligation process, you should carefully choose your backbone and insert - these both must have compatible cut sites for restriction enzymes that allow your insert to be placed into the backbone in the proper orientation.
Both the plasmid (blue, backbone) and the DNA sequence of interest (green, insert) are cut with restriction enzymes to generate compatible overhangs that allow them to bind. Overview of the restriction cloning process.
0 kommentar(er)
