Which step is used to increase uptake of plasmid DNA into competent cells during transformation?

Which step is used to increase uptake of plasmid DNA into competent cells during transformation?

• A. Incubation at high temperature
• B. Chemical treatment with calcium chloride followed by heat shock
• C. Centrifugation and resuspension
• D. Gel electrophoresis

Answer: (B)

To get plasmid DNA into cells, you need to make the cell envelope temporarily permeable and then push the DNA inside with a quick stimulus. The chemical treatment with calcium chloride coats the cells with Ca2+ ions, which helps DNA overcome the repulsion between negatively charged DNA and the cell surface and brings the DNA close to the membrane. Following this, a brief heat shock creates a sudden temperature change that drives the DNA into the cytoplasm through transient pores in the membrane. This combination—chemical conditioning plus a short heat pulse—significantly increases the efficiency of DNA uptake. Other options don’t achieve this dual effect. Incubating at a high temperature alone would likely damage or kill cells and doesn’t promote DNA entry. Centrifugation and resuspension are routine handling steps and don’t enhance uptake. Gel electrophoresis separates DNA, not transform cells.

To get plasmid DNA into cells, you need to make the cell envelope temporarily permeable and then push the DNA inside with a quick stimulus. The chemical treatment with calcium chloride coats the cells with Ca2+ ions, which helps DNA overcome the repulsion between negatively charged DNA and the cell surface and brings the DNA close to the membrane. Following this, a brief heat shock creates a sudden temperature change that drives the DNA into the cytoplasm through transient pores in the membrane. This combination—chemical conditioning plus a short heat pulse—significantly increases the efficiency of DNA uptake.

Other options don’t achieve this dual effect. Incubating at a high temperature alone would likely damage or kill cells and doesn’t promote DNA entry. Centrifugation and resuspension are routine handling steps and don’t enhance uptake. Gel electrophoresis separates DNA, not transform cells.