Principle of Insertional Inactivation- When cloned DNA is inserted into a gene it inactivates its functioning. Sequence/ insert cloned into vectors within genes of antibiotic resistance, leads to loss of function.
1. Host cells (E. coli cells) with recombinant pBR322 plasmid are first plated on solid media (agarose containing) with the antibiotic ampicillin (assume that the insert has been ligated within the tetracycline resistance gene). Colonies from every single cell plate having the plasmid will develop overnight
2. A petri plate containing solid media with antibiotic tetracycline is kept carefully under aseptic conditions (Laminar flow hood)
3. A circular piece of velvet or velvet paper is aligned and pressed onto the colony containing ampicillin plate (master plate). With the same alignment it is pressed onto the tetracycline plate. Overnight only colonies not containing the insert will grow while due to insertional inactivation no colonies will grow which have the insert. The colonies which have the insert can easily be scored off by comparing the two plates.