Pre-treatment is generally not necessary for a single 0.5 mm punch in a 20 µL reaction or a single 1 mm punch in a 50 µL reaction. However, when using more or larger punches, they can be pre-treated to avoid possible inhibition of PCR.

To pre-treat, place a single punch in a PCR tube. Add 50 µL of nuclease-free water and heat to 50°C for 5 minutes. Remove the supernatant with a pipette tip and use the washed punch in the PCR reaction.