Preparation of Competent Cells Using Calcium Chloride - Amrita University

Summary

The procedure for preparing competent cells using calcium chloride involves making bacterial cells more receptive to DNA. This is achieved by suspending the cells in a calcium chloride solution, creating small holes in the cell walls. The process includes steps such as growing the bacterial culture, harvesting the cells, washing them in ice-cold solutions, and finally resuspending them in calcium chloride. The prepared competent cells are ready for transformation or can be stored at -70°C for future use. This method is essential for experiments requiring plasmid DNA uptake.

Takeaways

• 😀 Competent cells have altered cell walls that make them more receptive to DNA uptake.
• 😀 To make bacteria competent, their cells are treated with a solution containing calcium chloride.
• 😀 Materials required for preparation include LB broth, culture plates, calcium chloride, magnesium chloride, and various lab equipment.
• 😀 The process begins with inoculating a single bacterial colony into LB broth and incubating overnight at 37°C.
• 😀 After overnight incubation, the bacterial culture is transferred to a larger volume of LB broth and incubated for an additional 3 hours.
• 😀 The bacterial cells are then harvested by centrifugation at 4100 rpm for 10 minutes at 4°C.
• 😀 The cell pellets are resuspended in ice-cold MgCl2-CaCl2 solution and centrifuged again.
• 😀 After centrifugation, the cells are resuspended in 0.1M ice-cold calcium chloride solution.
• 😀 The competent cells are aliquoted into separate vials and kept on ice.
• 😀 Competent cells can be used immediately for transformation or stored at -70°C for later use.

Q & A

What are competent cells?

Competent cells are bacterial cells that have been treated to have permeable cell walls, allowing them to take up foreign DNA more easily.

Why is it necessary to make bacterial cells 'competent'?

Bacterial cells must be made competent to efficiently take up foreign DNA, such as plasmids, for applications like transformation in genetic experiments.

How are bacterial cells made competent in this procedure?

Bacterial cells are made competent by suspending them in a solution with high calcium concentration, which causes small holes to form in their cell walls, allowing DNA to pass through.

What role does calcium chloride (CaCl2) play in preparing competent cells?

Calcium chloride helps to destabilize the bacterial cell wall, allowing DNA to enter the cells more easily during transformation.

Why is it important to use ice-cold solutions during the preparation of competent cells?

Ice-cold solutions help to stabilize the cells and slow down metabolic processes, which enhances the efficiency of the transformation process and helps maintain the cells in a viable state.

What is the purpose of centrifuging the cells during the procedure?

Centrifugation is used to pellet the bacterial cells, separating them from the medium so that they can be resuspended in fresh solutions like MgCl2-CaCl2 and CaCl2 for further processing.

What is the purpose of using LB broth in the preparation of competent cells?

LB broth is a nutrient-rich medium that supports bacterial growth. It is used to grow the bacterial culture before making the cells competent.

What is the significance of using a shaking incubator at 37°C?

The shaking incubator provides aeration and maintains the culture at an optimal temperature for bacterial growth, ensuring that the bacteria grow rapidly and reach the desired density.

Can the competent cells be frozen after preparation?

Yes, competent cells can be frozen at -70°C for future use. This preserves their ability to take up DNA when thawed and used in transformation experiments.

How do you ensure that the competent cells are properly aliquoted for use in transformation?

After preparing the competent cells, 100 microlitres of cells are aliquoted into each vial and kept on ice. This ensures that each vial contains the proper number of cells for subsequent transformation reactions.