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Competent E. coli Cell Preparation Using the Calcium Chloride Method

Product Manager:Harrison Michael



1. Overview:

The calcium chloride method is a well-established laboratory procedure used to prepare competent E. coli cells, which are capable of taking up foreign DNA through transformation. The process involves treating cells with a solution containing calcium chloride, which enhances the permeability of the bacterial cell wall to DNA.

 

2. Materials:

E. coli strain: Typically, strains like DH5α, XL1-Blue, or Top10 are used for transformation.

LB Broth: For growing E. coli cultures.

Calcium Chloride (CaCl₂) Solution (0.1M): Freshly prepared and kept on ice.

Sterile Glycerol (10% v/v): For storage of competent cells.

Sterile distilled water: For washing cells.

Ice-cold sterile pipette tips and tubes: For handling cells.

 

3. Protocol:

Step 1: Inoculation and Growth

• Inoculate 5 mL of LB broth with a single colony of E. coli and incubate overnight at 37°C with shaking (200-250 rpm).

 

Step 2: Expansion of Culture

• Transfer 1 mL of the overnight culture into 100 mL of fresh LB broth in a 500 mL flask.

• Grow the culture at 37°C with shaking until it reaches an OD600 of 0.4-0.6 (mid-log phase), typically 2-3 hours.

 

Step 3: Harvesting the Cells

• Chill the culture on ice for 15-30 minutes to slow down cellular processes.

• Transfer the culture to sterile, pre-chilled centrifuge tubes.

• Centrifuge at 4,000 rpm for 10 minutes at 4°C to pellet the cells.

 

Step 4: Washing the Cells

• Discard the supernatant carefully without disturbing the cell pellet.

• Resuspend the cell pellet gently in 20 mL of ice-cold 0.1M CaCl₂ solution using a pipette.

• Incubate the suspension on ice for 30 minutes.

• Centrifuge again at 4,000 rpm for 10 minutes at 4°C and discard the supernatant.

 

Step 5: Preparing Competent Cells

• Resuspend the cell pellet in 10 mL of ice-cold 0.1M CaCl₂ solution.

• Incubate on ice for another 30 minutes to enhance competency.

• Optional: Add sterile 10% glycerol to a final concentration of 10% for long-term storage.

 

Step 6: Aliquoting and Storage

• Aliquot the competent cell suspension into sterile, pre-chilled microcentrifuge tubes (typically 50-100 µL per tube).

• Snap-freeze the aliquots in liquid nitrogen or a dry ice/ethanol bath.

• Store the competent cells at -80°C for long-term use.

 

4. Quality Control:

• Transformation Efficiency: Test the competency of cells by transforming with a known plasmid (e.g., pUC19) and plating on selective media. Calculate the transformation efficiency by counting the number of colonies.

• Viability Check: Plate a small volume of cells before and after the competency procedure to ensure the cells remain viable.

 

5. Notes:

• Temperature Sensitivity: Keep all solutions and materials ice-cold throughout the procedure to maximize cell competency.

Glycerol Addition: Glycerol is optional but recommended if the competent cells are to be stored for an extended period.

• Sterility: Ensure all steps are performed under sterile conditions to avoid contamination.

 

This method provides a reliable way to produce competent E. coli cells suitable for DNA transformation experiments in the laboratory. Aladdin provides high-quality biochemical reagents related to competent preparation, and has a wide range of biological products to help the output of high-quality experimental results.

 

For more information, visit our website:

Aladdin:https://www.aladdinsci.com/

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