Wild-type l phage has a genome approximately 48,000 bp in size.  One commonly used l vector, Charon 4A, can be digested with EcoRI to generate four fragments, two large (arms) and two small central fragments (stuffers).

Since the stuffer fragments are not necessary for lytic growth of the phage, they can be separated by gel electrophoresis and removed.  Appropriate segments of genomic DNA having EcoRI sites at either end (~ 4000 bp) can be inserted to replace the stuffers.


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Since EcoRI has a six-nucleotide recognition sequence, it normally cuts DNA once every 4000 bp.  However, by shortening incubation time and reducing enzyme concentration (partial digestion), it is possible to produce restriction fragments averaging 5 X 400 = 20,000 bp.  l vectors are packaged in infective phage particles, which will only form if the DNA is approximately the same size as wild-type l phage DNA.

At the same time, E. coli infected with mutant strains of l phage DNA incorporated in their genomes (lysogens), are induced to enter the lytic phase.  This is temperature sensitive (ts), since the lysogen is inactive at 30o C, but leaves the chromosome at 42o C.  However, each mutant lysogen lacks a gene for a capsid protein (capsomere), so capsomeres accumulate in the cytoplasm, but viruses are not assembled.  The cells are then lysed and the phage products from each harvested as "packaging extracts".

Capsomeres can spontaneously assemble around DNA in-vitro, so by mixing the packaging extracts and recombinant l phage/chromosomal DNA, viable phage particles are produced.

Phages are spread on plates having a confluent "lawn" of E. coli.  After incubation at 37 oC, plaques or zones of lysis appear in the plates, containing millions of copies of the 20,000 bp genomic DNA insert.  Since cultures are inoculated to contain ~ 10,000 plaques, it is possible to represent at least three copies of the entire human genome on 15 plates.