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- Thaw competent cells on ice.
- Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
- Add 50 μl of competent cells to the DNA and mix gently by pipetting up and down.
- Incubate on ice for 30 minutes.
- Heat shock for 2 minutes at 37°C, chill on ice for 5 minutes.
- Add 950 μl of room temperature media and incubate at 37°C for 1 hour.
- Spread 100 μl onto the appropriate solid medium.
- Incubate overnight at 37°C.