General Cloning Protocol
"Cloning" is the term used in molecular biology for the insertion of another organism's gene into a target host organism (eg. taking the Green Fluorescent Protein (gfp) gene from the A. victoria jellyfish and putting it in E. coli to get E. coli to glow green). Though this sounds simple, multiple in vitro steps are involved that are not always simple in execution. One should remember that each of these in vitro steps is in essence a chemical reaction, which is subject to the environmental conditions, quality of reagents, and theoretical yields. If the yield for any of these reactions is too low, then we are unlikely to get the desired product (our gene of interest inserted into our target plasmid) at the end of our chain of reactions. These steps are generally as follows:
- PCR'ing or cutting the gene of interest from a heterologous template.