Download Cloning

Cloning
Using Plasmid Vectors
Vector
= a molecule used as a vehicle to carry
foreign DNA into a host cell
Simplest vector = plasmid
Features of Plasmids
Size
Functions encoded
Structure
Nomenclature:
R-plasmids
ColE1
Now standardised
Why are plasmids suitable
cloning vectors?
Generally do not kill host cell
Relatively easy to purify
Can be made small
Why not use naturally occurring
plasmids?
Too large
No selectable markers
Lack of unique recognition sequences
for restriction enzymes
Replication
Plasmid = replicon
Requires an origin of replication (oriV)
What is required?
Functions of the ori region
Host range
Narrow vs Broad
Copy number
Features of Plasmid Cloning
Vectors
Contain an oriV that allows for high copy
number, may have narrow (pUC) or broad
(R) host ranges
Small – why is this an advantage?
Selectable Genes
Unique restriction sites
May have additional features such as mob
sites, RNA polymerase promoters, etc.
pBR322
1973-1978
Bolivar and Rodriguez derivative 322
4.36 Kb; ~16 copies per cell
Narrow host range
Encodes resistance to ampicillin and to
tetracycline
How can we tell if plasmid
contains DNA of interest?
Insertional inactivation
Use BamHI site in Tetr gene for cloning
Transform
Plate cells on?
Amp – why not Tet?
Confirm by replica-plating on?
Tet – what do you expect to see?
Early 1980s – pUC series
Features of pUC Plasmids
Small
Very high copy number
No insertional inactivation
Blue-White Selection
Vector contains first 146 aa of the bgalactosidase gene (lacZ) (a-peptide)
MCS embedded within this region
Blue-White Selection Continued
Host cell encodes carboxy terminal
portion of lacZ
Neither host nor plasmid encodes for
entire protein
Together produce enzyme that can
cleave Xgal to produce blue precipitate
What if foreign DNA inserted into
MCS?
Foreign DNA will contain a termination
codon in the same reading frame as the
a-peptide
No a-peptide therefore no bgalactosidase and no blue coloured
colonies
pGEM series
Additional Features
Origin of DNA replication from ss filamentous
phage such as F1 or M13
Phagemid vector
T7 and SP6 promoters
www.promega.com
Variations on the pGEM
theme
pGEMT vectors (Hengen, 1995)
Make use of unique property of Taq
Facilitates easy cloning of PCR products how?
Limitations?