In Vivo Cloning

Question 1

Outline of in vivo cloning

Answer 1

• insertion of isolated gene into a vector (plasmid)
• transformation: transfer of plasmid vector containing the isolated DNA fragment into a suitable host (bacteria)
• identification of successfully transformed bacteria (contain the plasmid and DNA fragment)
• replica plating
• cloning of successful host cells

Question 2

What is a gene marker?

Answer 2

• used to identify which plasmids have taken up the DNA fragment
• the gene marker is disrupted if the DNA fragment is present- the gene won’t be expressed
• examples: antibiotic resistance, adding a fluorescent protein, making an enzyme with an identifiable product
• if these are lost, the DNA fragment has been taken up (success)

Question 3

Relplica plating

Answer 3

• all bacteria grown on master plate
• bacteria which grow on ampicillin but not tetracycline plate have been transformed successfully with plasmids
• select these colonies from the master plate