The blunt-ended cDNA is made compatible with the EcoRI sites through joining on synthetic linkers that contain EcoRI sites; these are joined covalently through the action of DNA ligase. Upon treatment with EcoRI, which creates a single sticky site at either end of the cDNA, the insert and the cleaved l phage can be annealed. Note that because adjacent phage can also join together via their cos sites, concatamers result. These are broken down into amounts of DNA required to create single phage genomes during the packaging of this DNA to form viable phage particles. These phage particles can be propagated through plaque formation on a bacterial lawn.
d. Library screening
The final step involves library screening, facilitating selection of the particular clone from the library that you are interested in.
Plating out the phage on to bacterial lawns is the first step of the screening process. Replicas are produced by placing absorbent paper, nitrocellulose or nylon membranes, over the agar plates in such a way that the pattern of plaques on the original plate is maintained. Each replica is then incubated with an appropriate nucleic acid probe that is labelled in some way (eg. radiolabelling) so as to facilitate selection. If the probe is a radiolabelled fragment of DNA, the location of the positive plaques may be detected through exposure to X-ray film, this process being known as autoradiography.
Typically, these will be grown up and plated out at least twice more so as to ensure that one is dealing with monoclonal material.
