Bacterial Transformation

These flashcards encapsulate key concepts, definitions, and procedures regarding bacterial transformation as discussed in the lecture notes.

What are the five steps of natural transformation in bacteria?

Development of competence, binding of DNA, incorporation of DNA, forming a pre-integration complex, integration into the host genome.

What is the role of CaCl2 in bacterial transformation?

Divalent cations promote DNA binding to membranes and reorganize lipopolysaccharides and proteins in the outer membrane.

What factors influence the efficiency of plasmid transformation?

Type of plasmid DNA, state of the host cell, preparation of cells, exposure to DNA, and expression conditions.

What is the purpose of using a selectable marker in plasmids?

To allow for the identification of successfully transformed cells.

What is electro-transformation and its advantage?

A method that uses high voltage to make cell membranes temporarily permeable to DNA, resulting in 100-1000 times higher transformation efficiency.

What should be done before adding DNA to competent E. coli cells?

Cells should be thawed on ice and allowed to recover prior to DNA addition.

Explain the process of preparing competent cells using CaCl2.

Cells are grown, washed with CaCl2, incubated on ice, then subjected to heat shock.

What is the transformation protocol using CaCl2?

Centrifuge the cells, resuspend in MgCl2 solution, keep on ice, and freeze immediately after resuspension.

What is the typical procedure for a practical transformation experiment?

Thaw competent cells, add ligation mix, incubate on ice, perform heat shock, add LB, and plate onto selective media.

What is the dilution factor used in calculating transformation efficiency?

Transformation Efficiency = CFU / 1 mg DNA.