METHODS IN GENE CLONING

1) Isolation

Target DNA and plasmid are isolated / extracted.

2) Cleave / Cut

Target DNA and plasmid are 'cut' at the specific site using the same restriction endonuclease.

3) Insertion

The gene of intrest is inserted into the cut plasmid. DNA ligase is used to bind the sticky ends together. Thus two pieces of DNA are joined together. Recombinant DNA is formed.

4) Transformation & amplification

  • Recombinant DNA is transferred into host cells, whereby the host cells are usually the E.coli bacteria.


  • The transformed bacteria is then cultured in a culture medium containing chloram phenicol. Chloramphenicol stimulates plasmid amplification. After plasmid amplification, the bacterial cells are placed onto the nutrient agar in a petri dish. Each bacterium will divide and form a visible colony.

5) Screening

  • The screening process is used to identify the transformed bacteria which have sucessfully taken up the recombinant DNA.
  • Blue-white screening : Bacteria are placed in a culture medium containing antibiotic (penicillin) and x-gal.

Steps involving in Polymerase Chain Reaction (PCR)

1) Denaturing

  • The DNA is heated at 95 degree celcius for a few seconds.
  • The double-stranded DNA is separated into its single stranded components (single-stranded DNA).
  • Both strands act as template.

2) Annealing

  • The mixture is cooled to 55 degree celcius and incubated for a few minutes.
  • Primers will anneal to the ends of the single-stranded DNA.

3) Elongation / Extension

  • Temperature is raised again to 72 degree celcius for about 1 minute.
    -This allows Taq polymerase to catalyse the formation of new strands of DNA.