IGEM:British Columbia/Protocols/Colony PCR
< IGEM:British Columbia | Protocols
- BioBrick PCR primers (G1004, G1005)
- Taq polymerase
- Buffer, Mg2+, dNTPs
- Agar plate for indexing
- PCR tubes
- sterile toothpick/pipet tip/innoculating loop
- Make master mix of primers and other PCR components
- Add Taq polymerase.
- Aliquot 10uL per PCR tube.
- Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
- Run PCR:
- 94°C for 2 mins
- 94°C for 30 secs
- 56°C for 30 secs
- 72°C for 1 min per kb of expected product, rounded up to nearest kb
- Repeat steps 2-4 24 times.
- 72°C for 3 times longer then step 4 (i.e for a expected product of 1.8 kb, step 4 will be 2 min long, step 6 will be 3*2 = 6 min long
- 4°C hold
- Verify PCR products on an agarose gel.