Colony PCR

This Summer, we did colony PCR with Homemade Taq and Comercial Taq, so we have two different protocols

Colony PCR with Homemade Taq

Mix (per reaction)

• 2.0µL 10x Taq Buffer
• 0.4 µL 50mM MgCl2
• 0.4µL 10mM dNTPs
• 0.8µL 10µM Primer Forward
• 0.8µL 10µM Primer Reverse
• 0.4µL 10x diluted Homemade Taq
• 12.2µL ddH₂O (deionized water)

Protocol

1. Aliquot 4µL of ddH₂O into a 0.2mL PCR tube
2. Using a pipette tip, gently touch one of your colonies and mix the cells into the water
3. To expose the DNA through cell lysis, place the tubes in the thermocycler at 95°C for 10 min
4. Add 17µL of your mix in each tube
5. Set the following program in the thermocycler:
• Stage 1 (1 cycle): 95°C for 3 min
• Stage 2 (36 cycles): 95°C for 30 sec, 55°C for 1 min, 72°C for 1min/kb
• Stage 3 (1 cycle): 72°C for 10 min
• Hold at 4°C
6. Run the products in a agarose gel

Colony PCR with Comercial Taq

Mix (per reaction)

• 5.0µL 10x Taq Buffer
• 1.5 µL 50mM MgCl2
• 1.0µL 10mM dNTPs
• 1.0µL 10µM Primer Forward
• 1.0µL 10µM Primer Reverse
• 0.2µL 10x diluted Homemade Taq
• 40.0µL ddH₂O (deionized water)

Protocol

1. Aliquot 4µL of ddH₂O into a 0.2mL PCR tube
2. Using a pipette tip, gently touch one of your colonies and mix the cells into the water
3. To expose the DNA through cell lysis, place the tubes in the thermocycler at 95°C for 10 min
4. Add 49.7µL of your mix in each tube
5. Set the following program in the thermocycler:
• Stage 1 (1 cycle): 95°C for 3 min
• Stage 2 (36 cycles): 95°C for 30 sec, 55°C for 1 min, 72°C for 1min/kb
• Stage 3 (1 cycle): 72°C for 10 min
• Hold at 4°C
6. Run the products in a agarose gel