PCR Protocol


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Materials Needed :

  1. DNA Template – The DNA you wish to amplify.
  2. Primers – Short single-stranded sequences of DNA that are complementary to the target DNA region.
    • Forward primer
    • Reverse primer
  3. DNA Polymerase – A heat-stable enzyme (e.g., Taq polymerase) that synthesizes new DNA strands.
  4. Nucleotide Mix (dNTPs) – A mixture of the four nucleotides (dATP, dTTP, dCTP, dGTP) required for DNA synthesis.
  5. Buffer Solution – Provides the optimal conditions for the DNA polymerase activity, including MgCl₂.
  6. Thermal Cycler – A machine that cycles the temperature during PCR.

Steps of the PCR Protocol :


1. Prepare the PCR Reaction Mixture:

  • DNA Template: 1-10 ng
  • Forward Primer: 0.1-0.5 µM
  • Reverse Primer: 0.1-0.5 µM
  • dNTPs (each): 0.2 mM
  • PCR Buffer (with Mg²⁺): 1X final concentration
  • DNA Polymerase (e.g., Taq polymerase): 0.5-1.0 U
  • Add water to bring the total volume to 25-50 µL

2. Denaturation (Initial Step):

  • Heat the reaction mixture to 94-98°C for 2-5 minutes to denature the DNA, breaking the hydrogen bonds between the strands.

3. Amplification Cycle (repeated 25-40 times):

  • Denaturation: Heat to 94-98°C for 20-30 seconds to denature the double-stranded DNA.
  • Annealing: Lower the temperature to 50-65°C (depending on primer Tm) for 20-40 seconds to allow the primers to bind to the DNA template.
  • Extension: Raise the temperature to 75-80°C (for Taq polymerase) for 1 minute per kilobase of DNA to allow DNA polymerase to synthesize the new DNA strand.

4. Final Extension:

  • After the last cycle, hold at 72°C for 5-10 minutes to ensure the full extension of any incomplete products.

5. Hold:

  • The reaction is stored at 4°C or -20°C for long-term storage of the amplified DNA.

Example PCR Conditions:

  • Initial Denaturation: 94°C for 5 minutes
  • Cycle (x35):
    • Denaturation: 94°C for 30 seconds
    • Annealing: 55-60°C for 30 seconds
    • Extension: 72°C for 1 minute
  • Final Extension: 72°C for 5 minutes

Post-PCR:

After amplification, the PCR product can be analyzed using gel electrophoresis to confirm the size of the amplified DNA fragment. The product can also be used for downstream applications like cloning, sequencing, or further analysis.

This is a basic outline, and protocols can be adjusted based on the specific requirements of the experiment, such as the type of DNA template and primers used.