Some Additional Tools for the Molecular Biologist - Insect Molecular Genetics - page 217

likelihood of producing complete genomic libraries. Efficiency of infection of
E. coli with “packaged” DNA can be 10 6 recombinants per microgram of vector
DNA, an increase in efficiency over transfection by nearly 2 orders of magnitude.
In vitro packaging involves several steps and specific conditions ( Figure 6.4 ).
One critical condition is the size of the inserted DNA. The amount of exogenous
DNA inserted into the vector must be regulated carefully: the cos sites must be
separated by DNA that is ≈ 78-105% of the length of the wild-type chromosome.
In an insertion vector, only 14kb of DNA, or less, can be cloned. In a replace-
ment vector, up to 22 kb of DNA can be inserted. In replacement vectors, a pair of
restriction sites flanks the nonessential central region of the phage DNA called the
“stuffer region.” When the stuffer region is excised and the insert DNA is ligated
into this region, a DNA molecule is produced that can be packaged efficiently.
In vitro packaging requires the following components: 1) the DNA mole-
cules to be packaged, 2) high concentrations of phage-head-precursor protein,
3) proteins that participate in the packaging process, and 4) phage tails. These
Figure 6.4 In vitro packaging of cloned DNA in λ involves providing a protein head and tail precur-
sor. The DNA is cut at the cos sites, and if the DNA is ≈ 50 kb, it will be packaged in the head. The
complete phage is then used to infect E. coli and amplify the recombinant molecule.