July 21th 2016

Transformation: competent DH5⍺ cells with ligation products BB1, BB2 and BB3

Objectives

The objective is to transforme competent DH5⍺ cells with the ligation products BB1, BB2 and BB3 to create a stock of transformed bacteria.

Materials

6 aliquots of NEB DH5⍺ Competent E.coli (C2287)

Plasmid DNA : Ligation product BB1, BB2 and BB3 (from the 20/07/16)

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

Protocol

Experimental conditions achieved :

We need 20 LB+Cm plates + 11 LB plates

Transformations protocol:

haw 2 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.
Add the 20 µL / 30 µL plasmid DNA to the cell mixture.
Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex.
Place on ice for 30 min. Do not mix.
Heat shock at exactly 42°C for 45 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 1h at 250 rpm.
Warm selection plates to 25°C.
Mix the cells thoroughly by flicking the tubes and inverting.
Spread the corresponding volume onto each plate.
Incubate all the plates O/N at 37°C.

Results (obtain the 22/07)

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 dish without antibiotic.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).

Obtained results:

We obtained expected results for petri-dishes plated with bacteria transformed with BB1 and BB2, but there is no colonies for petri-dishes plated with bacteria transformed with BB3.

Interpretation

The transformation worked for BB1 and BB2. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore in order to know if bacteria incorpore the correct plasmids BB1 and BB2. A new digestion, ligation and transformation is necessary for BB3.