Set up a PCR master mix without contaminating it

Reinforce CLSI unidirectional PCR order. Template is not added until the clean master mix is aliquoted, the no-template control is pipetted first in the dirty area, and post-PCR tubes do not re-enter the clean room.

1. Work in the clean room or dead-air box with dedicated pipettes.
2. Prepare the master mix with no template present.
3. Aliquot mix into reaction tubes.
4. Move sealed mix aliquots to the separate template area.
5. Pipette the no-template control first.
6. Add template last.
7. Seal tubes before they leave for the thermocycler.
8. Never open amplified product in the clean room.