Cutting DNA

1
Cutting DNA

• Restriction endonucleases (restriction enzymes)
• sticky ends
• blunt ends
• Nomenclature
• EcoRI
• E genus (Escherichia)
• co species (coli)
• R strain
• I of enzyme

2
Blunt Sticky ends
3
Pasting DNA

• Complementary ends (sticky ends) H-bond
• Ligase forms phosphodiester bond to seal strands
  together.

4
A plasmid vector for cloning

• Contains an origin of replication, allowing for
  replication independent of hosts genome.
• Contains Selective markers Selection of cells
  containing a plasmid
• twin antibiotic resistance
• blue-white screening
• Contains a multiple cloning site (MCS)
• Easy to be isolated from the host cell.

5
Plasmid vectors
6
Expression vector
7
How to clone DNA

• Isolation of cloning vector (bacterial plasmid)
  gene-source DNA (gene of interest)
• Insertion of gene-source DNA into the cloning
  vector using the same restriction enzyme bind
  the fragmented DNA with DNA ligase
• Introduction of cloning vector into cells
  (transformation by bacterial cells)
• Cloning of cells (and foreign genes)
• Identification of cell clones carrying the gene
  of interest

8
Screening of the clone

• The medium in this petri dish contains the
  antibiotic Kanamycin
• The bacteria on the right contain Kanr, a plasmid
  that is resistant to Kanamycin, while the one on
  the left has no resistance
• Note the difference in growth

9
Blue/White Color Screening
lacZ
10
Selecting Colonies with Recombinant Plasmids