1. Reagents

We always use a high-fidelity DNA polymerase (such as Q5, Phusion, or Pfu) to amplify a DNA fragment for molecular cloning to minimize the chance of introducing mutations. However, for genotyping or other applications that do not require high-fidelity, Taq and its enhanced derivatives are perfectly fine. Our current favorite is the Q5 Hot Start High-Fidelity 2X Master Mix.

2. Procedure

1. Prepare PCR reactions. We usually use the 8-tube PCR strips.

   Stock	µL for 50 µL	×5	×10	×15
   2× Q5 master mix	25	125	250	375
   Water	18	90	180	270
   Primer 1	2.5	12.5	25	37.5
   Primer 2	2.5	12.5	25	37.5
   Template (10-20 ng/µL)	2	10	20	30

2. Typical cycling conditions:

   • 98°C, 2 min

   • 30× cycles of:
     • 98°C, 10 sec
     • 55-60°C, 15 sec
     • 72°C, 10 sec/kb for short fragments, ~30 sec/kb for long fragments]

   • 72°C, 2 min

   • 12°C, hold