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 PCR reagents to a tube
- Setup additional tubes as needed- always include a positive and negative control
- Add tubes to PCR machine, and run with program below
- Run PCR product out on a gel
Reagents for PCR
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
PCR program
- 94°C 5 min
- 94°C 30s
- 55°C 30s
- 72°C 1min
- Loop to #2 for 35 cycles
- 72°C 2min
- 10°C indefinitely