Standard PCR

Master mix assembly, thermal cycling, and a gel check.

5 steps · Nucleic acids · PCR

1. 1. Assemble the master mix on ice

   Thaw components on ice, vortex briefly and spin down. Assemble the mix in a chilled tube; add polymerase last.

2. 2. Aliquot master mix into the strip tubes

   Dispense the mix into each reaction tube, then add template last to avoid carry-over.

3. 3. Initial denaturation

   Hold at 98 °C to fully denature the template before cycling.

4. 4. Thermal cycling

   30 cycles of 98 °C denature / 60 °C anneal / 72 °C extend. Set the anneal temperature from your primer Tm.

5. 5. Check products on a gel

   Run an aliquot alongside a ladder and confirm a single band at the expected size.