Bacterial transformation

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Bacterial transformation of chemically competent cells 
Javier Álvarez, Yachay Tech University 
1. Label sterile microfuge tubes 
2. Always include a minus-DNA negative control and a positive control of a 
known plasmid 
3. Thaw on ice the frozen competent cells (or use them after being made) 
4. Add 0.1 ml of cells per tube 
5. Add 2 µl of plasmid DNA (up to 100 ng DNA) and mix gently 
6. Incubate on ice for 25 min (24 h incubation increases the efficiency). 
Have ready a water bath or heat block at 42 oC for the next step 
7. Heat shock the cells for 40”-2’ at 42 oC, then bring them back to ice 
8. Transfer each transformation mixture to a test tube with 1 ml of LB or 
SOC medium. Place them on a roller or a gently shaker at 37 oC for 1 h 
9. Plate 100 - 200 µl on the appropriate antibiotic plate and incubate at 
37 oC 
Materials and Reagents 
• Ice, small pieces 
• Sterile tips 
• Competent cells, thawed on ice 
• 2 plasmids: the experimental and the positive control 
• Water bath or heat block at 42 oC 
• 3 Test tubes with LB broth or SOC medium 
• LB + antibiotic plates, also one LB plate as extra control 
• 37 oC incubator