Cloning simply means making an exact copy of DNA. There are three main types of cloning:
Organismal cloning involves making a copy of all nuclear DNA to make an identical organism; this is what most people think of when they think of cloning. If you had a clone, he or she would share exactly the same genome, like an identical twin.
Therapeutic cloning is used to generate stem cells from your own skin cells. Someday, this technology might help treat degenerative diseases such as Parkinson’s.
Molecular cloning involves making copies of DNA fragments. The DNA fragment you want to clone is inserted into bacteria. Then, as the bacteria multiply, they make super-pure copies of this DNA.
For your BioPREP project, you will be doing Molecular Cloning, or using bacteria to clone the DNA fragments you amplified in the PCR step.
Bacteria are useful for molecular cloning because they’re easy to grow, multiply quickly, and have a genome that has already been deciphered. Bacteria, unlike eukaryotes, can have additional DNA in structures called plasmids. Plasmids are small pieces of DNA that float outside the main bacterial genome in the cytoplasm of the cell. Plasmids used for cloning are called vectors.
The first step in cloning is to attach your PCR product to the vector. The TOPO kit is designed so you simply combine the vector and your PCR product and they ligate.
Transformation is the process in which bacteria take up DNA, in this case, your vector+PCR product. The combination of salt and heat begins this process, though the exact mechanism is unknown.
How do you know if the bacteria are transformed and have taken up the vector? The TOPO vectors come with a gene for antibiotic resistance. This is why special agar plates with antibiotic (the selective plates) were prepared for the bacteria. The bacteria that haven’t taken up the vector or aren’t expressing the protein adequately will die and the ones which contain the vector (and your PCR product) will live and form colonies.