In vivo

• In vivo cloning


  1. Cut DNA fragment using restriction endonuclease enzymes

  2. Add promoter region

  3. Add terminator region

  4. Insert DNA fragment into vector

  5. Get vector into host cell
• Sticky ends


  Overhangs created by staggered cuts in double-stranded DNA by restriction endonuclease enzymes
• Plasmids


  Circular loops of DNA in bacterial cells, not part of main bacterial DNA, often contain few genes
• Inserting DNA fragment into plasmid vector

  1. Cut plasmid with same restriction endonuclease as used on DNA fragment

  2. Combine DNA fragment and plasmid due to complementary sticky ends

  3. Use DNA ligase to glue DNA fragment into plasmid
• Transformation


  Getting plasmid vector into host bacterial cell
• Transformation


  1. Make cell membrane permeable by adding calcium ions and heat shock

  2. Plasmid enters cytoplasm of host cell
• The gene inserted into the host cell will be expressed, meaning the protein it codes for will be made
• The purpose of in vivo cloning is to amplify DNA fragments that have been isolated