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 (often carried out by enzymes), which is subject to the environmental conditions (salt concentration, temperature), quality of reagents (how much DNA, presence of dNTP's, etc), 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. Therefore, we should always seek to optimize the yield of desired product for each of our reactions; this simply means use the HIGHEST quality reagents in the most OPTIMAL conditions you can.
Here is a highly generalized set of steps in cloning:
- Production of the linear insert. This is usually done by PCR'ing out the desired fragment or cutting it out of a plasmid that already has the desired insert.
- Cutting the insert and target vector with appropriate endonucleases. We use endonuclease enzymes to cut specific sequences of DNA.