Typical PCR experiment

A typical PCR experiment is carried out under the following conditions:

  • 1X polymerase buffer
  • 0.2 mM each primer
  • 200 mM each dNTP
  • 2.5 U DNA polymerase
  • DNA template*

* 100-250 ng for mammalian genomic DNA and 20 ng for linearized plasmid DNA per 50 ml reaction mix.

The experiment consists of three phases:

  1. Initial denaturation. 3-5 min at 92-94°C.
  2. Amplification. This phase consists of 25 to 40 cycles of:
    • Denaturation. 10-30 sec at 92-94°C.
    • Annealing. 10-60 sec at the annealing temperature (50-68°C).
    • Extension. 60 sec * product length (in kb) at 72°C.
  3. Final extension. 5-10 min at 72°C.

Experimental conditions for a typical PCR experiment are described in the following protocol.

PCR with longer DNA templates

For PCR with longer DNA templates (3-7 kb) better results are obtained by lowering the extension tempearture to 68°C. No or lower amounts of PCR product is obtained at 72°C caused by a higher level of template depurination at this temperature.