Cloning strategies - Principles of Gene Manipulation - page 88

Polylinker Polylinker

Bam H I Bam H I

Sal I Eco R I Eco R I Sal I

red + gam +

AB

λ EMBL3A

Target high molecular
weight genomic DNA
(1) Partial Sau 3A I digest
(2) Phosphatase

Sal I Sal I

Digest with Bam H I
and Eco R I

AB Bam H I red + gam +

Bam H I

Sau 3A I Sau 3A I Sau 3A I Sau 3A I

Eco R I Eco R I

HO OH HO OH

Left arm
Polylinker
oligonucleotide

Right arm
Polylinker
oligonucleotide

Sau 3A I Sau 3A I Sau 3A I Sau 3A I

Isopropanol precipitation
Polylinker oligonucleotide
not precipitated

HO OH HO OH

Sau 3A I Sau 3A I

AB Bam H I Bam H I

HO OH

Size fractionation not necessary

Eco R I

Left arm Right arm

Ligate, mix

AB ( Bam H I ) ( Bam H I )

+

Recombinant phage
DNA concatemers

Left arm Right arm

Sal I Sal I

Packaging in vitro

Plate on
P2 lysogen
sup +

Recombinant
(Spi - ) phage
plaques

Fig. 6.3 Creation of a genomic DNA library using the phage- vector EMBL3A. High-molecular-weight genomic DNA is partially

digested with Sau 3AI. The fragments are treated with phosphatase to remove their 5

λ

phosphate groups. The vector is digested
with Bam HI and Eco RI, which cut within the polylinker sites. The tiny Bam HI/ Eco RI polylinker fragments are discarded in the
isopropanol precipitation, or alternatively the vector arms may be purified by preparative agarose gel electrophoresis. The vector
arms are then ligated with the partially digested genomic DNA. The phosphatase treatment prevents the genomic DNA fragments
from ligating together. Non-recombinant vector cannot re-form because the small polylinker fragments have been discarded.
The only packageable molecules are recombinant phages. These are obtained as plaques on a P2 lysogen of sup + E. coli . The Spi −
selection ensures recovery of phage lacking red and gam genes. A sup + host is necessary because, in this example, the vector carries
amber mutations in genes A and B . These mutations increase biological containment, and can be applied to selection procedures,
such as recombinational selection, or tagging DNA with a sup + gene. Ultimately, the foreign DNA can be excised from the vector
by virtue of the Sal I sites in the polylinker. ( Note : Rogers et al . (1988) have shown that the EMBL3 polylinker sequence is not
exactly as originally described. It contains an extra sequence with a previously unreported Pst I site. This does not affect most
applications as a vector.)

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