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plasmids with inserts. Since replica plating gives an identical pattern of colonies on

both sets of plates, it is straightforward to recognise the colonies with inserts, and to

recover them from the ampicillin plate for further growth. This illustrates the impor-

tance of a second gene for antibiotic resistance in a vector.

Although recircularised plasmid can be selected against, its presence decreases the

yield of recombinant plasmid containing inserts. If the digested plasmid is treated with

the enzyme alkaline phosphatase prior to ligation, recircularisation will be prevented,

since this enzyme removes the 5^0 phosphate groups that are essential for ligation.

Links can still be made between the 5^0 phosphate of insert and the 3^0 hydroxyl of

plasmid, so only recombinant plasmids and chains of linked DNA fragments will be

formed. It does not matter that only one strand of the recombinant DNA is ligated,

since the nick will be repaired by bacteria transformed with these molecules.

The valuable features of pBR322 have been enhanced by the construction of a series

of plasmids termed pUC (produced at the University of California) (Fig. 6.13). There is

an antibiotic resistance gene for tetracycline and origin of replication forE. coli.In

addition the most popular restriction sites are concentrated into a region termed the

multiple cloning site(MCS). In addition the MCS is part of a gene in its own right and

codes for a portion of a polypeptide calledb-galactosidase. When the pUC plasmid has

been used to transform the host cellE. colithe gene may be switched on by adding the

inducer IPTG (isopropyl-b-D-thiogalactopyranoside). Its presence causes the enzyme

b-galactosidase to be produced (Section 5.5.5). The functional enzyme is able to

hydrolyse a colourless substance called X-gal (5-bromo-4-chloro-3-indolyl-

b-galactopyranoside) into a blue insoluble material (5,5^0 -dibromo-4,4^0 –dichloro

indigo) (Fig. 6.14). However if the gene is disrupted by the insertion of a foreign

Ampicillin
resistance gene

pUC18

2686 bp

ORI

ApR

lacZβ-galactosidase gene

Multiple cloning site

(MCS) polylinker

Control regions for lacZ

Origin of replication

lacI

HindIII

SphI

PstI

HincII

AccI

SalI

BamHI

XmaI

SmaI

KpnI

SacI

EcoRI

Fig. 6.13Map and important features of pUC18.

210 Recombinant DNA and genetic analysis