In order to detect which of the many plaques on the plate contains the rearranged Ig gene, the plate is probed with the same J probe used to detect rearrangement in the Southern blots. First of all, the plaques are transferred to a nitrocellulose filter. Finally, the filters are pre-hybridized, hybridized, washed, and exposed exactly as for the Southern blot. If there are any positive areas, the plaque can be selected from the original plate and grown, and the DNA is isolated. Once the DNA has been isolated from a positive phage, it is recloned into a plasmid vector.
Plasmids are easier to propagate and store than phages.
To reclone the insert, the phage DNA is cut with the same restriction enzyme used to cut the genomic DNA to make the library and re-ligated into the plasmid that has been cut with the same enzyme, and transformed into bacteria. Each sequencing reaction can read about 200-300 basepairs. To do this we make a restriction map of the clone. In order to determine which restriction fragment contains your gene of interest we have several options. We can do a Southern on the DNA with all the various restriction enzymes and probe it with the J probe.