Gene Cloning

56 Gene Cloning
(a) (b) (c)
Ap r
lacZ'
MCS
Ap r
pUC18
Ap r
Ap r resistant lac negative
(d)
Ap r
lacZ'
Cut with restriction enzyme
Mix and ligate
Ap r and lac positive
Figure 3.14a In a cloning experiment using insertional inactivation of lacZ′ in
pUC18 the plasmid and genomic DNAs a) are cut with a restriction enzyme
which cuts in the multiple cloning site (MCS), and then are mixed and ligated b).
In successful recombinants, lacZ′ will be non-functional because it has been
interrupted by the cloned fragment c); if the vector recircularizes the lacZ′ gene
will be functional and β-galactosidase will be produced.
Recombinant clones can be identified
because they are lac minus, there is
no need for replica plating
Agar contains ampicillin,
X-gal and IPTG
Figure 3.14b Recombinant clones can be identified because they do not express
β-galactosidase, and hence they will be white on plates containing X-gal and
IPTG. Colonies containing recircularized vector will be blue.
the rest of the enzyme (Box 3.4). Both the α peptide and the rest of the β-
galactosidase enzyme are required for activity. The multiple cloning site in
pUC18 is in the middle of the lacZ′ gene so cloning into it will result in
insertional inactivation of the gene and the resulting clones will be unable
to break down X-gal. These recombinant clones will be white, and are easily
distinguishable from those containing the recircularized vector, which
will be blue (Figure 3.14a and b).