Team:Groningen/protocols/Transformation EC

E. coli transformation protocol

Materials:

• LB plates with selection markers (antibiotics, inducers,…)
• LB broth
• Eppendorf tubes (preferably 2 ml)
• Spreaders
• DNA to be transformed

Reaction procedure:

1. Prepare plates with the correct antibiotic selection marker.
2. Prechill the eppendorf tubes on ice.
3. Remove a tube of frozen competent E. coli (DH5α) cells from the storage (-80°C freezer) and thaw it on ice (about 5 minutes). Mix the cells by gently flicking the tube. Avoid excessive pipetting, as the competent cells are extremely fragile.
4. Carefully transfer 50 μl of cells into each tube prepared in Step 2.
5. Add 10 μl ligation reaction to the competent cells.
6. Gently flick the tubes to mix and place them on ice for 30 minutes.
7. Heat-shock the cells for 60 seconds in a water bath at 42°C (do not shake and make sure the content of the tube is all at the bottom).
8. Immediately return the tubes to ice for 2 minutes.
9. Add 1 ml LB broth (RT) to the reaction mixture.
10. Incubate at 37°C for 1 hour with shaking (~150 rpm).
11. Centrifuge the tubes at 14000 rpm for 1 min.
12. Empty the tube until about 200 μl supernatant is left.
13. Resuspend the pellet in the supernatant.
14. Plate 200 μl of each transformation culture on LB agar plates with the correct resistant marker.
15. Incubate the plates overnight (16–24 hours) at 37°C.
16. Afterwards the plates can be stored at 4°C.