Team:ETH Zurich/Experiments
Experimental: Protocols, Methods and Material
Polymerase Chain Reaction - for Construction
Phusion ® High-Fidelity DNA Polymerase
Composition:
| Component | 50 µl Reaction |
| Phusion HF Buffer (5x) | 10 µl |
| dNTPs (10 mM) | 1 µl |
| Forward Primer (10 µM) | 2.5 µl |
| Reverse Primer (10 µM) | 2.5 µl |
| Template DNA | variable |
| DMSO (optional) | up to 3% |
| Nuclease-free water | up to 50 µl |
| Phusion Polymerase | 0.5 µl |
Thermocycling Conditions:
| Temperatur |
Time |
| 98°C | 30 seconds |
| 98°C 45-72°C 72°C |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
| 72°C | 5-10 minutes |
Kapa Hifi Hotstart Ready Mix
| Component | 50 µl Reaction |
| Master Mix (2x) | 25 µl |
| Forward Primer (10 µM) | 2.5 µl |
| Reverse Primer (10 µM) | 2.5 µl |
| Template DNA | variable |
| Nuclease-free water | up to 50 µl |
Thermocycling Conditions:
| Temperatur |
Time |
| 95°C | 3 minutes |
| 98°C 55-75°C 72°C |
20 seconds 15 seconds 15-60 seconds per kb |
| 72°C | 1 minute per kb |
Polymerase Chain Reaction - for Analysis
Solis BioDyne FirePol® DNA Polymerase
Composition:
| Component | 8 x 20 µl Reaction (+ 10 µl backup) |
| FIREPol DNA Polymerase | 0.85 µl |
| MgCl2 (25 mM) | 10.2 µl |
| Reaction Buffer B (10x) | 17 µl |
| dNTPs (10 mM) | 3.4 µl |
| Forward Primer (10 µM) | 3.4 µl |
| Reverse Primer (10 µM) | 3.4 µl |
| Template DNA | 1 µl |
| Nuclease-free water | 125 µl |
Thermocycling Conditions:
| Temperatur |
Time |
| 98°C | 5 minutes |
| 95°C 50-72°C 72°C |
30-60 seconds 30-60 seconds 1 minute per kb |
| 72°C | 5-10 minutes |
Thanks to the sponsors that supported our project:
