Jonesma Lab:Protocols:colony
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Colony PCR
- Take a colony and resuspend in 5 μL dH2O
- Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony
- 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
- Setup additional tubes as needed- always include a positive and negative control
- 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
- Run PCR product out on a gel