How to Make Competent Cells: Protocols and Tips

Bacterial transformation is one of the most commonly performed techniques in molecular biology. This process transfers exogenous DNA (e.g. plasmids, gene deletion cassettes, etc.) into a host cell. Once within the cell, the DNA can then be incorporated into the genome, replicated, used to produce proteins, and more. However, transformation can be an inefficient process that requires methodological tricks to move the DNA past the bacterial membrane(s) and cell wall and make the bacteria “competent” for DNA uptake.

What Are Competent Cells?

Competent cells are bacterial cells that are capable of taking up and incorporating exogenous DNA from their surroundings. Many bacterial species can naturally uptake DNA from the environment. However, the most commonly genetically transformed lab bacteria, Escherichia coli, cannot.

To overcome a lack of natural competence, E. coli can be treated by a number of procedures to render it able to take up DNA. Typically, researchers use chemical (and heat shock) or electroporation means to transform, although other methods exist. The process of making competent cells introduces pores into the cell membrane which allow them to uptake extracellular DNA more readily. Once competent cell preparation is complete, the E. coli cells are ready for DNA transformation. Transformation is the process by which foreign DNA is introduced into host cells, resulting in specific genetic modifications. This process is integral for applications such as gene cloning and recombinant protein expression.

What is Transformation Efficiency?

Transformation efficiency is commonly used to describe how well competent cells take up DNA. This value is described as the number of colony-forming units (cfu) produced by transforming 1 µg of plasmid DNA into a given number of competent cells. For example, an ideal efficiency would be 10⁸ cfu/µg of DNA. For a detailed explanation of transformation efficiency and how it can be calculated, please visit another blog post that covers everything you need to know about bacterial transformation.

High-quality plasmid DNA should be used, as contaminants or low DNA concentrations can undermine the transformation process. ZymoPURE plasmid purification kits are the fastest way to purify high-yield, endotoxin-free plasmid DNA that is ideal for transformations.

The transformation efficiency is affected by a variety of factors including the genotype of target cells, plasmid size, supercoiled vs. relaxed DNA, growth phase of cells at time of collection, and method of transformation. Given the variety of potential confounding factors, special care must be taken to ensure successful transformation experiments.

Protocol for Competent Cell Preparation

Specialized equipment and materials are needed to prepare chemically competent E. coli in-house, as indicated below.

Required Materials:

1. E. coli cells (typically DH5α or similar strains)
2. LB (Luria-Bertani) broth and agar plates
3. Calcium chloride (CaCl₂)
4. Ice
5. Sterile microcentrifuge tubes
6. Centrifuge
7. Incubator set to 37 °C
8. Pipettes and tips
9. Sterile distilled water

The recommended guidelines for producing chemically competent cells may vary depending on several factors, such as the E. coli strain used and the growth rate of the culture. However, most traditional methods for preparing chemically competent cells consist of the following steps.

Step 1: Inoculation and Growth of Cells (3-4 hours)

• Commence by inoculating a single colony of E. coli into 5-10 ml of LB broth.
• Incubate the culture at 37 °C with shaking until it reaches an optical density (OD600) of about 0.4–0.6.

Step 2: Chilling Cells on Ice (10-15 minutes)

• Transfer the culture to a sterile centrifuge tube.
• Place the tube on ice for 10-15 minutes to cool down the cells.

Step 3: Centrifugation and Resuspension of Cells (30 minutes)

• Centrifuge the culture at 4,000 x g for 10 minutes at 4 °C.
• Carefully decant and discard the supernatant.
• Gently resuspend the cell pellet in an equal volume of cold, sterile 100 mM calcium chloride (CaCl₂) solution.
• Incubate on ice for 30 minutes, gently inverting the tube every 5-10 minutes.

Step 4: Repeating Centrifugation and Resuspension (30 minutes)

• Centrifuge the cells again at 4,000 x g for 10 minutes at 4 °C.
• Carefully remove the supernatant, leaving the cell pellet.
• Resuspend the pellet in a smaller volume (usually 100-200 μl) of cold 100 mM CaCl₂ solution.

Step 5: Aliquoting and Freezing (15-20 minutes)

• Divide the competent cells into small aliquots (usually 20-100 μl) and store them in sterile microcentrifuge tubes.
• Flash-freeze the aliquots in liquid nitrogen and store them at -80 °C until needed.

Thus, preparing chemically competent cells in-house requires a large time investment, proper equipment and materials, and technical expertise. The process can be long and tedious, requiring several temperature-sensitive incubation steps that must be completed properly to achieve efficient transformations downstream. As an alternative to traditional procedures, the Mix & Go! E. coli Transformation Kit and Buffer Set simplifies the process of competent cell preparation by employing a fast 3-step protocol that can be completed in less than 45 minutes.

4 Tips for Making Competent Cells

Competent cells should be prepared meticulously and stored correctly to ensure they can be used for highly efficient transformation. The list below can help you steer clear of problems when making chemically competent cells using traditional in-house procedures.

Tip #1: Keep Them COLD!

The process of making chemically competent cells is challenging due to the need for the cells to stay cold. This is crucial because the cells are so sensitive and fragile while they are being made competent. Keeping the temperature low helps to avoid cell death during processing.

This means using a cold room if possible and performing the centrifugations at 0–4 °C. Pre-cooled microcentrifuge tubes and racks should also be used and kept on dry ice before use. Doing this allows the cells to freeze faster, important especially when aliquoting the cells. Flash-freezing in liquid nitrogen before storing cells at -80 °C can help maintain cell competency for an extended period.

Keeping the cells cold is even important during the culturing process. Lower temperature growth (18–33 °C) increases transformation efficiencies. Due to this, we recommend using ZymoBroth growth medium, as it can promote efficient growth even at lower temperatures.

Tip #2: Handle Cells Carefully

Gentle resuspension of cells prevents damage and maximizes transformation efficiency. Additionally, complete removal of the CaCl₂ solution after the second centrifugation step is critical to prevent excess salts from inhibiting subsequent transformations.

Tip #3: Prepare Freezer Space

Competent cells need to be stored at -80 °C. The process of making the cells competent makes them very fragile – likely to rupture and die. This means that storing at -20 °C can dramatically reduce transformation efficiency. After just 24 hours of storage at -20 °C, cells can lose up to 90% of the transformation efficiency.

Tip #4: Prepare Reagents and Labware

Making chemically competent cells can be a long and tedious process with several lengthy incubations. The process requires use of sterile growth media, glassware, and processing reagents. This requires preparation of all reagents and labware prior to starting the procedure.

The Easiest Way to Get Competent Cells

An alternative option to making competent cells is using commercial strains, which are premade competent cells. This eliminates many of the hassles associated with this time-consuming process and ensures optimal transformation efficiency, as the strains have already been measured and validated. The best option for rapid and efficient transformation is the Mix and Go! Competent Cells. They have very high transformation efficiencies of up 10⁹ transformants per µg of plasmid DNA and bypass the conventional heat shock procedure to perform transformations in 20 seconds (for ampicillin resistance-based plasmids). Five premade strains including the popular DH5α strain are commercially available for a wide range of applications, from basic cloning to complex genome editing.

Competent cells are one of the most commonly used reagents within the lab and having the right cells is crucial for any successful transformation. Whether you choose to purchase or make competent cells, the technical support team at Zymo Research is available to help you through the process.