Objectives

The objective is to transform competent DH5⍺ cells with BBX4, BBC5 and BBC1 in order to create a stock of transformed bacteria.

Materials

• 3 aliquots of DH5⍺ competent cells (from the 20/09/16)
• Plasmid DNA:
• • BBX4: “RBS - mRFP-his” (RBS - mRFP), 717bp , from the iGEM plate 5: 1F (BBa_K1491009)
  • BBC5: “GFP reporter for RHS of library test constructs”, from the iGEM plate 4: 11L (BBa_I1340)
  • BBC1: “Test Device 1” (Preference - RBS - GFP - Term ; J23101.B0034.E0040.B0015)
• Petri dish LB+Cm: Cm concentration = 25 µg/mL

Protocol

Experimental conditions realized:

Transformation Protocol:

1. Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice.
2. Add the 1 µL (200/300ng) plasmid DNA to the cell mixture.
3. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.
4. Place on ice for 30 min. Do not mix.
5. Heat shock at exactly 42°C for 45 s. Do not mix.
6. Place on ice for 5 min. Do not mix.
7. Pipette 250 µL of room temperature SOC into the mixture.
8. Place at 37°C for 1h at 250 rpm.
9. Warm selection plates to 25°C.
10. Mix the cells thoroughly by flicking the tubes and inverting.
11. Spread the corresponding volume onto each plate.
12. Incubate all the plates O/N at 37°C.

Results (obtained on the 02/10)

Expected results:

Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
A bacterial lawn on the LB petri dishes without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results.

Interpretation

The transformations worked. Colonies incorpore the plasmid with the Chloramphenicol resistance gene, present in pSB1C3.