pGLO transformation

Question

Hi!

I did a lab about pGLO transformation, and in the instructions, it is said that the heat shock must last for exactly 50 seconds. But why is time so important here?

Answer 1

The key step in transformation is heat shocking the bacterial cells to allow changes in the permeability or structure of the cell membrane to allow the plasmid/gene to enter the cells. There’s a delicate balance here for several reasons. Heat must be transferred rapidly through the cells to deliver a sharp shock. If the heat shock step is done for too long or at the wrong temperature, either no colonies would be seen on transformation plates or there would be low transformation efficacy. This basically means that the gene/plasmid would not be expressed. This is actually a common error in many experiments and often is a cause of skewed results.

This is also why the recovery step done afterwards is so important. If the structure/permeability of the cell membranes is repaired too quickly, the plasmid won’t be expressed. Also, if you don’t have proper recovery of the colonies/cells, the plasmid again doesn’t have a chance to be expressed and/or grow.