How to Transform E. coli by Heat Shock

Synthetic Biology One
8 Sept 201711:02

Summary

TLDRThis video script outlines the heat shock transformation process for E. coli bacteria. It begins with thawing competent cells and taking an aliquot, followed by adding a small amount of plasmid DNA. The mixture is then heat shocked at 42°C for 30 seconds and allowed to recover on ice. Cells are then cultured in LB media at 37°C for recovery, before being plated on antibiotic-containing agar to select for successful transformations. The process results in colonies of transformed bacteria, showcasing the effectiveness of the heat shock method.

Takeaways

  • 🌡️ The video demonstrates the heat shock transformation method for E. coli, which involves preparing cells for the uptake of plasmid DNA.
  • ⏰ Cells are first thawed and kept on ice to maintain a cold environment, which is crucial for the transformation process.
  • 📊 Only a small volume of cells, 20 microliters out of 200, is needed for the transformation.
  • 💉 A tiny amount of plasmid DNA, half a microliter, is added to the cells for transformation, indicating a high concentration is used.
  • 🧊 The mixture of cells and plasmid is kept cold to ensure the plasmid sticks to the cells, potentially improving transformation efficiency.
  • 🕒 A waiting period of 5 to 10 minutes is suggested before the heat shock to allow the plasmid to bind to the cells.
  • 🔥 The heat shock step involves raising the temperature to 42 degrees Celsius for exactly 30 seconds to facilitate DNA uptake.
  • ❄️ After the heat shock, cells are immediately placed back on ice for 2 minutes to recover.
  • 🥫 Recovery of the cells is done in plain LB media, which is found to be effective for most applications, instead of enriched media like SOC.
  • 🚦 A recovery period of 15 minutes to an hour at 37 degrees Celsius allows cells to grow and express the antibiotic resistance marker on the plasmid.
  • 🌿 Cells are shaken during the recovery step to create optimal conditions for cell growth, using a 'sandwich method' to ensure thorough mixing.
  • 📝 Two plates are prepared for plating the cells, one with 200 microliters and one with 20 microliters, to increase the chances of obtaining the right number of colonies.
  • 🧪 Glass beads are used to spread the media evenly on the plates, preventing the absorption of bacteria and ensuring an even distribution.
  • 📆 The plates are incubated overnight to allow for bacterial colony formation, which can then be observed after approximately 16 hours of growth.

Q & A

  • What is the purpose of heat shock transformation in the script?

    -Heat shock transformation is a method used to introduce plasmid DNA into E. coli bacteria. The process involves a brief exposure to high temperatures to make the bacteria more receptive to take up the DNA.

  • How long do the E. coli cells need to thaw before use?

    -The cells need to thaw for about 10 minutes before they are ready to use for transformation.

  • Why is it important to keep everything cold during the transformation process?

    -Keeping everything cold helps to prevent the cells from growing and also protects the DNA from degradation by enzymes.

  • How much of the plasmid is added to the competent cells in the script?

    -Only half a microliter of the plasmid is added to the competent cells for successful transformation.

  • What is the significance of flicking the tube after adding the plasmid?

    -Flicking the tube helps to gently mix the cells with the plasmid without introducing too much shear force that could damage the cells.

  • Why is it recommended to let the cells sit for 5-10 minutes after mixing with the plasmid?

    -Allowing the cells to sit for 5-10 minutes gives the plasmid time to bind to the cells, which can improve transformation efficiency.

  • What is the temperature and duration for the heat shock step?

    -The heat shock step involves raising the temperature to 42 degrees Celsius for exactly thirty seconds.

  • Why do the cells need to be placed on ice after the heat shock?

    -Placing the cells on ice after the heat shock allows them to recover from the stress of the high temperature.

  • What type of media is used to recover the cells after the heat shock?

    -Plain LB (Luria-Bertani) media is used to recover the cells after the heat shock.

  • How long do the cells need to recover at 37 degrees Celsius?

    -The cells need to recover at 37 degrees Celsius for 15 minutes to an hour, depending on the plasmid and antibiotic resistance used.

  • Why are two plates prepared with different volumes of cells?

    -Preparing two plates with different volumes of cells increases the chances of getting a plate with the optimal number of colonies for counting and further analysis.

  • What is the purpose of using glass beads when plating the cells?

    -Glass beads are used to spread the media evenly around the plates without absorbing too many bacteria, resulting in a nice even distribution of bacteria on the plate surface.

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Ähnliche Tags
BiologyLab TechniqueE.coliTransformationHeat ShockPlasmidMicrobiologyProtocolLab SkillsScientific
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