Setting Up PCR Reactions Using Phusion PCR Master Mix
Carefully mic and centrifuge all tubes before opening to improve recovery. PCR reactions should be set up on ice.
50 µl reaction
20 µl reaction
|H2O||add to 50 µl||add to 20 µl|
|2x Phusion Master Mix||25 µl||10 µl||1X|
|Primer A *||x µl||x µl||0.5 µM|
|Primer B *||x µl||x µl||0.5 µM|
|Ttemplate DNA||x µl||xµl|
|(DMSO **, optional)||(1.5 µl)||(0.6 µl)||(3%)|
* The recommendation for final primer concentration is 0.5 μM, but it can be varied in a range of 0.2-1.0 μM if needed.
** Addition of DMSO is recommended for GC-rich amplicons. DMSO is not recommended for amplicons with very low GC% or amplicons that are >20kb.