Transformation of E. coli

Materials required

• E. coli DH5α (200 µl)
• Appropriate vector DNA
• Heat block
• Bucket of ice
• Pipettes
• Eppendorf tubes
• 1.5% agar plate containing appropriate antibiotics

Protocol

1. Thaw a 200 µl aliquot of E. coli DH4α and add the transforming DNA (10 ng of vector DNA in 10 µl).
2. Incubate on ice for 30 minutes.
3. Heat-shock the cells for 120 seconds at 42°C, and place on ice again for 3-4 minutes.
4. Add 1 ml of LB broth and incubate the cells at 37°C for 1-1.5 hr in a water bath with gentle shaking.
5. Plate 200 µl of transformed E. coli onto 1.5% agar plate containing the appropriate selection markers.
6. Incubate the plates overnight at 37°C.

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