PCR and Plasmid Cloning

📋 Course Outline

1. PCR Principle and Components
2. PCR Cycle and Amplification
3. Melting Temperature and Primers
4. PCR Reaction Optimization
5. Plasmid Vectors and Selection
6. Restriction Digestion and Cloning

📖 1. PCR Principle and Components

🔑 Key Concepts & Definitions

• Polymerase Chain Reaction : a fast and inexpensive technique that exponentially amplifies a selected target DNA molecule through repeated heating and cooling cycles

★ Must-know

• PCR applications include:

  • genotyping
  • cloning
  • mutation detection
  • medical diagnostics
  • sequencing

• A basic PCR requires:

  • a DNA template containing the target region
  • heat-resistant Taq DNA polymerase
  • two primers with free 3'OH groups
  • dNTPs
  • buffer
  • bivalent cations usually MgCl2
  • a PCR machine

Further detail

• PCR is credited to Kary Mullis in 1983, and he received the Nobel Prize in Chemistry with Michael Smith for this invention in 1993.

📖 2. PCR Cycle and Amplification

• Each PCR cycle consists of denaturation at 95°C, annealing at 50–65°C, and extension at 72°C.

• Under efficient reaction conditions, PCR can amplify one or a few starting copies of the target region to billions of copies.