Cloning Genes

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Cloning Genes

• Gene cloning amplifying a specific piece of DNA
  via a bacteria cell
• Cloning vector a replicating DNA molecule
  attached with a foreign DNA fragment to be
  introduced into a cell
• Has features that make it easier to insert DNA
  and select for presence of vector in cell.
• Origin of replication
• Antibiotic resistance gene
• Cloning site

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Cloning Genes

• Plasmid vectors
• Linkers synthetic DNA fragments containing
  restriction sites
• Transformation of host cells with plasmids
• Selectable markers are used to confirm whether
  the cells have been transformed or not.

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TA cloning of PCR product
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Bacterial transformation

• Use modified strain of E. coli
• Restriction system knocked out
• Recombination system limited
• LPS is truncated
• Pores are made in cell membrane by treatment with
  calcium chloride.
• Cells must be kept cold to prevent pores from
  closing

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Bacterial transformation

• DNA added to cells forms intimate interaction
  with membrane
• Briefly moved to 42 C to heat shock cells
• Stresses them and causes them to take up DNA