The following procedure is used to amplify DNA with Taq DNA polymerase in 50 ul of reaction mixtures. It can also be used for the ExTaq DNA polymerase (Takara Bio). However, since many DNA polymerases have different preferable conditions, manufacturers�f instructions should be consulted for other DNA polymerases, especially for high-fidelity enzymes.

1. Mix the following solutions on ice.

Template DNA 10x Taq DNA polymerase buffer dNTP mix (2.0-2.5mM each of 4dNTP) forward primer reverse primer H2O Taq DNA polymerase

usually less than 50 ng 5 ul 5 ul 10 pmol 10 pmol up to 49 ul 1 ul (0.5 unit)

2. Perform PCR following standard procedures (e.g., denaturation at 94 degrees, 40 sec; annealing at 55 degrees, 1 min; extension at 74 degrees, 1-5 min; 25-30 cycles).

* Typically, 1 ug of amplified DNA is obtained in 50 ul of the reaction mixture. The reaction volume can be reduced to 10 ul reducing the amount of each component and using a 0.2 ml-tube.