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 and quantity of reagents (how much cut DNA, presence of dNTP's, etc), and theoretical yields. If the yield for any of these reactions in our series is too low, then we are unlikely to get the desired end product (usually our gene of interest inserted into our target plasmid). 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. A linear piece of a desired DNA sequence can be obtained in many ways, including traditional PCR, Assembly PCR, cutting a piece out of an already existing vector (as in biobricks cloning), synthesis orders (through companies), primer extension reactions, and [primer annealing].
- Cutting the insert and target vector with appropriate endonucleases. We use restriction endonuclease enzymes to cut specific sequences of DNA. The ends of these cut pieces of DNA will then stick together if their sequences are complementary, allowing us to ligate linear pieces of DNA together in a specific order.
- Ligating the linearized vector and insert together. Here, we simply combine our insert and vector in a reaction with the enzyme DNA ligase, which covalently links free ends of DNA together.
- Transformation of the completed vector and screening. E. coli will take up DNA through its membranes under certain conditions (heat shock, electroporation), which allows us to put our finished DNA vector into the bacterium. We plate our transformants on selective media, which will allow only those cells to grow that have successfully taken up our vector.