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Standard polymerase chain reaction (PCR) protocol for amplification of DNA.


For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL premixed TAQ DNA polyermerase


  1. In a PCR tube, mix the components in the order they are listed above. Keep TAQ DNAP cold right up until it is added to the reaction volume. Mix gently and spin.
  2. Perform the following thermocycling program:
    1. 95 °C 5 min (initial melting)
    2. 95 °C 30 s (melting)
    3. TH 30 s (annealing)
    4. 72 °C 1 min for each 1 kb PCR product (elongation)
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min (final elongation)
    7. 12 °C hold (storage)
  3. Clean PCR product with PCR Purification Kit.



Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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Relevant papers and books

  1. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773


or instead, discuss this protocol.

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