NEBuilder® HiFi DNA Assembly Chemical Transformation Protocol
For use with NEBuilder® Hifi DNA Assembly Cloning Kit (NEB #E5520)and NEBuilder HiFi DNA Assembly Bundle for Large Fragments (NEB #E2623 )
- Thaw competent cells on ice.
- Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at 42°C for 30 seconds. Do not mix.
- Transfer tubes to ice for 2 minutes.
- Add 950 ul of room temperature SOC medium to the tube. When using NEB 10-beta or NEB Stable E. coli competent cells, add 950 ul of the NEB 10-beta/Stable Outgrowth Medium.
- Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Spread 100 μl of the cells onto the selection plates. Use Amp plates for positive control sample.
- Incubate overnight at 37°C.
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