Polymerase Chain Reaction (PCR) protocol

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Solutions/reagents:

  • Buffer
  • dNTPs
  • MgCl 2
  • DMSO
  • Upstream primer
  • Downstream primer
  • Template DNA
  • dH2O
  • DNA Polymerase

Equipment:

  • Thermocycler
  • Reaction tubes

Steps:

  1. For Promega GoTaq:
    Set up a reaction in reaction tube (1) as follows on ice:

    <thead></thead><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body>
     Initial concentrationFinal concentrationFinal volume per 50 µl reaction
    Buffer5X1X10 µl
    dNTPs1.25mM0.25mM10 µl
    MgCl 225mM2.5mM5 µl
    DMSO50%5%5 µl
    Upstream primer50pmol1pmol1 µl
    Downstream primer50pmol1pmol1 µl
    Template DNA??~0.5µg1 µl
    dH2ON/AN/A16.75 µl
    DNA Polymerase5u/?L1.25u0.25 µl

  2. For Roche High Fidelity:
    Set up a reaction in reaction tube (1) as follows on ice:

    <thead></thead><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body><tbody></body>
     Initial concentrationFinal concentrationFinal volume per 50 µl reaction
    Buffer10X1X5 µl
    dNTPs1.25mM0.25mM10 µl
    DMSO50%5%5 µl
    Upstream primer50pmol1pmol1 µl
    Downstream primer50pmol1pmol1 µl
    Template DNA??~0.5µg1 µl
    dH2ON/AN/A16.75 µl
    DNA Polymerase5u/?L2.5u0.5 µl

  3. An example program:
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 96°C, 5 mins
    This denatures any double stranded DNA.
    Thermocycling
    • No. of cycles: 25
    • Denature: 96°C, 1 min
    • Anneal: 55°C, 30 secs
    • Elongate: 72°C, 1 min
    Termination
    • Elongate: 72°C, 5 mins
    • Hold: 4°C, until removed from machine
    Final extension for incomplete strands and holding of sample at low temperature.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 15 mins

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