Jonesma Lab:Protocols:colony

Colony PCR

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1. Take a colony and resuspend in 5 μL dH₂O
2. Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony
3. Add the following reagents to a tube

2 μL 10x PCR buffer

2 μL 1/4 dNTPS

2 μL 20mM MgCl₂

6 μL dH₂O

1 μL 10μM Forward primer

1 μL 10μM Reverse primer

1 μL Taq

1. Setup additional tubes as needed- always include a positive and negative control
2. Add tubes to PCR machine, and run with following programme

1. 94°C 5 min 2. 94°C 30s 3. 55°C 30s 4. 72°C 1min 5. Loop to #2 for 35 cycles 6. 72°C 2min 7. 10°C indefinitely

1. Run PCR product out on a gel