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ANNETTE DEICHMANN AND KLAUS DEICHMANN
• pSP6/T7 (Melton 1984)
This vector shares many homologies with pUC. In addition, the plasmid
has been supplemented by 2 bacteriophage promotors flanking the MCR.
This allows the plasmid to be used as a cloning and in vitro transcription
vector.There are many other plasmids constructed to suit special tasks
(see catalogues of the main suppliers of products for molecular biology).
Bacteriophages
The use of plasmids is restricted to an insert size of up to about 5 kb. When
cloning larger fragments, bacteriophage should be used.
A.phage Lambda phage DNA is a 43 kb double-stranded DNA, that is introduced into
bacterial cells by transfection (Figure 3}. The DNA may be covalently incorporated into the bacterial genome and replicated with it as a prophage.
However, during the lytic cycle, hundreds of phage DNA copies are formed
and packaged by special enzymes into newly-formed phage particles.
Packaging enzymes and head and tail proteins are encoded by the phage
DNA. The bacterial cells are broken open (Iysis}, and phages are set
free. This mechanism also applies to other temperate phages such as phi
80, 82, 21, 434, P2 and 299.
• A.gtlO I A.gtll
These vectors are mainly used for cloning cDNA inserts after ligation to
EcoRI linkers. In A.gtlO the unique EcoRI cloning site lies in the ci (repressor) gene. This gene is inactivated by inserting DNA into the EcoRI
site. When using suitable host cells, infected cells bearing a nonrecombinant A.gtlO phage will form cloudy plaques whereas those bearing recombinant phage form clear plaques. Furthermore growth in E. coli carrying the hfl AlSOmutation will produce plaques only in insert-bearing
bacteriophage. A.gtlO is suitable for screeningwith nucleic acid probes. In
A.gtll (Young 1983} the cloning site lies within the lacZ a-peptide gene
(see M13 below). This allows unique EcoRI expression of cloned genes as
ß-galactosidase fusion proteins and screening with antibody probes.
Furthermore, discrimination between insert-bearing and insert-free
phage can be performed with a color indicator. When grown on plates
containingX-Gal, recombinant phage plaques are clear, whereas non-recombinant phage plaques appear blue (mechanism see M13 below).
ANNETTE DEICHMANN AND KLAUS DEICHMANN
• pSP6/T7 (Melton 1984)
This vector shares many homologies with pUC. In addition, the plasmid
has been supplemented by 2 bacteriophage promotors flanking the MCR.
This allows the plasmid to be used as a cloning and in vitro transcription
vector.There are many other plasmids constructed to suit special tasks
(see catalogues of the main suppliers of products for molecular biology).
Bacteriophages
The use of plasmids is restricted to an insert size of up to about 5 kb. When
cloning larger fragments, bacteriophage should be used.
A.phage Lambda phage DNA is a 43 kb double-stranded DNA, that is introduced into
bacterial cells by transfection (Figure 3}. The DNA may be covalently incorporated into the bacterial genome and replicated with it as a prophage.
However, during the lytic cycle, hundreds of phage DNA copies are formed
and packaged by special enzymes into newly-formed phage particles.
Packaging enzymes and head and tail proteins are encoded by the phage
DNA. The bacterial cells are broken open (Iysis}, and phages are set
free. This mechanism also applies to other temperate phages such as phi
80, 82, 21, 434, P2 and 299.
• A.gtlO I A.gtll
These vectors are mainly used for cloning cDNA inserts after ligation to
EcoRI linkers. In A.gtlO the unique EcoRI cloning site lies in the ci (repressor) gene. This gene is inactivated by inserting DNA into the EcoRI
site. When using suitable host cells, infected cells bearing a nonrecombinant A.gtlO phage will form cloudy plaques whereas those bearing recombinant phage form clear plaques. Furthermore growth in E. coli carrying the hfl AlSOmutation will produce plaques only in insert-bearing
bacteriophage. A.gtlO is suitable for screeningwith nucleic acid probes. In
A.gtll (Young 1983} the cloning site lies within the lacZ a-peptide gene
(see M13 below). This allows unique EcoRI expression of cloned genes as
ß-galactosidase fusion proteins and screening with antibody probes.
Furthermore, discrimination between insert-bearing and insert-free
phage can be performed with a color indicator. When grown on plates
containingX-Gal, recombinant phage plaques are clear, whereas non-recombinant phage plaques appear blue (mechanism see M13 below).