Colony PCR

Materials

Pick a single colony and place in 20µL of H20.
Cell lysis:
- Incubate 10 min @ 98°C
- Freeze 10 min @ -80°C
- Vortex for 2-5 min
In a PCR tube mix:
- 10µL of 2X Phusion Mastermix
- 1µL of DNA template (lysate
- XµL of Primer 1
- XµL of Primer 2
- 7µL of Nuclease free H2O
Run PCR:
- Initial Denaturation: 30s @ 98°C
- Denaturation: 10s @ 98°C
- Annealing: 30s @ Y°C
- Elongation: 30s per kb @ 72°C
- Reapeat steps 2-4, 25 to 35 times
- Final Elongation: 10 min @ 72°C
- Final Hold: ∞ @ 4°C

For Biobrick primers (VF2 and VR) from James Brown we used the following values:
- X = 1µL
- Y = 65°C
Datasheet for the 2X Phusion Mastermix