UA Biophysics:Protocols:Transformation of Competent Cells

TRANSFORMATION OF COMPETENT CELLS PROTOCOL

1. Place the competent cells on ice
2. Add the DNA (Plasmid) to the eppendorf containing the competent cells and pipette gently in order to mix them well. (1 uL of DNA or Plasmid is sufficient!!)
3. Leave the eppendorf on ice for 30min.
4. Incubate the cells at 42°C during 30 seconds
5. Put the cells on ice during 2min.
6. Add 1mL of LB at room temperature
7. Incubate during 1h at 37°C in a shaker. (Timing can be reduced to 45min)
8. Take 100-300uL of the cells and grow them in a petri dish with the corresponding antibiotic. (Be sure to spread the volume through the whole surface of the petri dish!...in the Biophysics lab we use some sterile glass beads for this part)
9. Place the petri dish in a 37°C incubator
10. Store the remaining cells in the eppendorf in a 4°C freezer (Just in case!!)

    If nothing grows on the petri dish, then take out the eppendorf that was stored in the 4°C freezer, and then grow (and spread) a bigger volume in another petri dish (with the corresponding antibiotic)

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