July 17th 2016

Transformation: NEB competent DH5⍺ cells with pSB1C3-RFP

Objectives

To transform competent DH5⍺ cells with pSB1C3-RFP that will be used as a backbone for the creation of biobricks.

Materials

1 aliquot of NEB DH5⍺ Competent E.coli

Plasmid DNA: "RFP Coding Device", BBa_J04450, from the iGEM plate 6-12P (concentration: 200-300 pg/µL)

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

Protocol

Resuspension of pSB1C3-RFP:

We use the protocol given by the iGEM and availablehere

With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
Pipette 10 µL of H2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 min to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.

Experimental conditions achieved

We need 3 LB+Cm plates + 2 LB plates

Transformation protocol

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.
Add 1 µL of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 min. Do not mix.
Heat shock at exactly 42°C for 30 s. Do not mix.
Place on ice for 5 min. Do not mix.
Pipette 250 µL of room temperature SOC into the mixture.
Place at 37°C for 60 min at 250 rpm.
Warm selection plates to 25°C.
Mix the cells thoroughly by flicking the tube and inverting.
Spread the corresponding volume onto each plate.
Incubate all the plates O/N at 37°C.

Results (obtained on the 16/07)

Expected results

Some red colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed 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 petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results.

Interpretation

The transformation worked. Colonies contain the plasmid with the Chloramphenicol resistance gene, present in pSB1C3, and the RFP gene, producing Red Fluorescent protein.