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/