Objectives

To test the competency of the DH5⍺ cells done on the 05/06/16 with the stock of pSB1C3-RFP plasmid.

Materials

Plasmid DNA : pSB1C3-RFP at 200 pg/µL

DH5⍺ competent cells (See 05/06/16)

Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

1. Thaw 1 tube of DH5α competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into 2 transformation tubes on ice.

2. Add 1 µL (200 pg) of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 min. Do not mix.

4. Heat shock at exactly 42°C for 30 s. Do not mix.

5. Place on ice for 5 min. Do not mix.

6. Pipette 950 µL of room temperature SOC into the mixture.

7. Place at 37°C for 60 min at 250 rpm.

8. Warm selection plates to 25°C.

9. Mix the cells thoroughly by flicking the tube and inverting.

10. Spread 100 µL of each dilution (100%,10%,1%) onto a selection plate.

11. Pellet the remaining cells (except for the control), discard mostly all the supernatant and resuspend the remaining cells in 100 µL of LB. Spread 100 µL onto a selection plate.

12. Incubate all the plates O/N at 37°C.

Results

Expected results :

Some colonies on the petri dishes LB+Cm plated with 1% of transformed bacteria, more with 10%, a lot with 100% and a bacterial lawn with the pellet.
A bacterial lawn is expected on the LB petri dishes (+ control).
No colony is expected on the LB+Cm petri dish plated with no plasmid (- control).
All obtained colonies are expected to be red because of the RFP gene that codes for a red fluorescent protein.

Obtained results:
We obtained expected results.

Interpretation

The transformation worked, as we obtained satisfying results. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. The DH5α cells are therefore competent.