PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit

Protocol

1. Set up the appropriate reactions on ice:

   Component	20 µl Reaction	50 µl Reaction	Final Concentration
   Nuclease-free water	13.6 µl	34 µl
   5X Phusion HF Buffer	4 µl	10 µl	1X
   10 mM dNTPs	0.4 µl	1 µl	200 µM
   Primers*	1 µl	2.5 µl	0.2 µM
   Control Template DNA	0.8 µl	2 µl	0.02 ng/µl
   Phusion DNA Polymerase	0.2 µl**	0.5 µl	0.02 U/µl

   *Reaction can be set up using either the 1.3 or 10 kb primer set
   ** Dilute polymerase with 1X reaction buffer to avoid pipetting errors
   
   
2. Recommended cycling conditions for the 1.3 kb fragment using a 2-step protocol:

   Cycle step	Temp	Time
   Initial denaturation	98°C	1 Minute
   25 Cycles	98°C 72°C	5 Seconds 20 Seconds
   Final extension	72°C	10 Minutes
   Hold	4°C	∞

   
   Recommended cycling conditions for the 10 kb fragment using a 3-step protocol. Alternatively, this program can be used to amplify both fragments simultaneously.

   Cycle step	Temp	Time
   Initial denaturation	98°C	1 Minute
   25 Cycles	98°C 60°C 72°C	5 Seconds 15 Seconds 2 Minutes 30 Seconds
   Final extension	72°C	10 Minutes
   Hold	4°C	∞

   
   Note: Controls have been shown to work in a variety of conditions.