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Cloning

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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 ... – PowerPoint PPT presentation

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Title: Cloning


1
Cloning
  • Using Plasmid Vectors

2
Vector
  • a molecule used as a vehicle to carry foreign
    DNA into a host cell
  • Simplest vector plasmid

3
Features of Plasmids
  • Size
  • Functions encoded
  • Structure
  • Nomenclature
  • R-plasmids
  • ColE1
  • Now standardised

4
Why are plasmids suitable cloning vectors?
  • Generally do not kill host cell
  • Relatively easy to purify
  • Can be made small

5
Why not use naturally occurring plasmids?
  • Too large
  • No selectable markers
  • Lack of unique recognition sequences for
    restriction enzymes

6
Replication
  • Plasmid replicon
  • Requires an origin of replication (oriV)
  • What is required?

7
Functions of the ori region
  • Host range
  • Narrow vs Broad
  • Copy number

8
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.

9
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

10
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11
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?

12
Early 1980s pUC series
13
Features of pUC Plasmids
  • Small
  • Very high copy number
  • No insertional inactivation

14
Blue-White Selection
  • Vector contains first 146 aa of the
    b-galactosidase gene (lacZ) (a-peptide)
  • MCS embedded within this region

15
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

16
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 b-galactosidase and no
    blue coloured colonies

17
pGEM series
18
Additional Features
  • Origin of DNA replication from ss filamentous
    phage such as F1 or M13
  • Phagemid vector
  • T7 and SP6 promoters

19
www.promega.com
20
Variations on the pGEM theme
  • pGEMT vectors (Hengen, 1995)
  • Make use of unique property of Taq
  • Facilitates easy cloning of PCR products - how?
  • Limitations?
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