How to Make Chemically Competent E. coli

Summary

In this video on synthetic biology, the process of preparing competent E. coli cells is demonstrated. The steps include preparing two calcium chloride solutions, inoculating and growing E. coli, and diluting the culture for optimal competency. After centrifugation, cells are resuspended in calcium chloride and glycerol solutions and kept cold to maintain competency. The cells are then aliquoted into smaller tubes for immediate use or storage. Careful temperature control is emphasized throughout the procedure to ensure cell viability and efficiency during transformation.

Takeaways

• 🧪 Preparing competent cells involves making two solutions: 100 mM calcium chloride and 100 mM calcium chloride with 15% glycerol.
• 🔬 Accurate measurement is not critical for these solutions; 'close is good enough' as they are forgiving.
• ❄️ Solutions should be stored on ice to maintain a temperature of 4 degrees Celsius to prevent cell lysis.
• 🌡️ The process starts with a single colony of E.coli to ensure genetic homogeneity and lack of contamination.
• 🚫 It's important to work with log phase E.coli for competent cell preparation as they are healthier and more competent.
• 🌡️ The culture is grown at 37 degrees Celsius with shaking until it reaches an OD of 0.5 or 0.6, indicating mid-log phase.
• 🧊 After centrifugation, cells are resuspended in calcium chloride solution and kept on ice to maintain low temperatures.
• 🕒 The cells are left on ice for at least four hours, or even overnight, to become competent.
• 🔄 A second round of centrifugation is performed, and cells are resuspended in calcium chloride with glycerol for transformation readiness.
• 🏗️ The final step involves aliquoting the cells into smaller tubes for convenient use in transformations, with each tube containing enough for multiple uses.

Q & A

What are the two solutions needed to prepare competent cells in synthetic biology?

The two solutions needed are a 100 millimolar solution of calcium chloride and another solution of 100 millimolar calcium chloride with 15% glycerol.

How much calcium chloride is required to prepare the solutions?

One and a half grams of calcium chloride is required for both solutions.

Why is it important to label the solutions near the top and what is the risk if not done so?

The solutions should be labeled near the top because they will be stored on ice, and if the labels get wet, they can fall off.

What is the purpose of adding water to the calcium chloride solution before the full 100 milliliters?

Adding water before the full volume allows room for the glycerol to be added later without exceeding the total volume.

Why is it recommended to be careful when pipetting glycerol?

Glycerol can be difficult to pipette and can coat the entire pipette if not done carefully, which can affect the accuracy of the measurement.

What is the significance of storing the prepared solutions on ice?

Storing the solutions on ice is crucial to maintain the cells at four degrees Celsius, preventing them from lysing if the temperature rises.

What type of E. coli strain is used to start the culture for making competent cells?

A wild type strain of E. coli, specifically mg 1655, is used, but other cloning strains like nab turbo or dh5 alpha could also be used.

Why is it important to start the culture from a single colony of E. coli?

Starting from a single colony ensures that the cells are genetically homogeneous and not contaminated.

What is the purpose of diluting the saturated E. coli culture into a fresh 50 ml culture?

Diluting the culture helps to use freshly growing log phase E. coli, which are the healthiest and most competent for transformation.

At what optical density (OD) should the E. coli cells be to proceed with the competent cell protocol?

The cells should be at an OD of 0.5 or 0.6, indicating they are in the mid-log phase.

How are the cells prepared for the second round of centrifugation in the competent cell protocol?

After the first round of centrifugation, the cells are resuspended in calcium chloride solution and left on ice for at least four hours. For the second round, they are resuspended in calcium chloride and glycerol solution.