Setting Up PCR Reactions Using Phusion PCR Master Mix
Introduction
Carefully mic and centrifuge all tubes before opening to improve recovery. PCR reactions should be set up on ice.
| Component | Volume/ 50 µl reaction |
Volume/ 20 µl reaction |
Final Conc. |
| 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.
