Question

after transforming E colo with the pGLO plasmid, you incubate the vials with LB broth for 30 min first. Explain what may happen if you skip this step.

Answer 1

The heat shock increases the permeability of the cell membrane to DNA. The duration of the heat shock is critical in the transformation experiments. The standard protocol for pGLO transformation of Escherichia coli is adding L-arabinose to LB medium at a concentration of 6 g L⁻¹ along with ampicillin at a concentration of 100 mg L⁻¹. To demonstrate the specificity of the interaction between sugars and the AraC protein, other carbohydrates can be added to the medium instead. E. coli that has been transformed with pGLO plasmid DNA are grown in the presence of arabinose undergone a 30-min incubation period following the addition of LB nutrient broth. This allows the transformed cells to recover and to express the ampicillin resistance protein β-lactamase so that the transformed cells survive on the ampicillin selection plates. This is why the vials contain transformed E. coli with the pGLO plasmids incubated 30 min first. If we skip this step the transformed cells cannot survive in ampicillin. The incubation of recovery culture can increase the transformation efficiency by more than 10-fold.