Heat Shock Transformation Protocol

Introduction

• Purpose: Enable bacterial cells to take up circular plasmid DNA.
• Application: Introduce genes of interest into bacteria for expression.

Transformation Overview

• Natural Transformation: Uptake of free genetic material from the environment by bacteria.
• Bacterial DNA Components:
  • Chromosomal DNA
  • Plasmid DNA

Plasmid DNA Uptake

• Some bacteria can uptake exogenous plasmid DNA when another bacterium dies.
• Gene Expression: Genes from foreign plasmids may be expressed, leading to protein production.

Artificial Transformation Methods

• Heat Shock Protocol
• Electroporation

Heat Shock Transformation Steps

1. Inducing Competence

   • Bacterial cells are made competent using calcium chloride treatment.

2. Preparation

   • Keep competent cells on ice for 10 minutes.

3. DNA Addition

   • Add plasmid DNA containing gene of interest to cells.
   • Incubate cell-plasmid mixture on ice for 30 minutes.

4. Calcium Chloride Role

   • Brings DNA close to bacterial outer membrane's lipopolysaccharides.
   • Calcium cations mask charges on DNA and lipopolysaccharides.

5. Heat Shock

   • Direct transfer from ice to a 42°C water bath.
   • Duration: 30 seconds to 1 minute.
   • Facilitates plasmid DNA entry into cytosol.

6. Post Heat Shock

   • Return cell-plasmid solution to ice.
   • Add growth medium and incubate at 37°C with agitation.
   • Purpose: Regenerate damaged cell membrane, allow cell division and gene expression.

7. Selection and Growth

   • Plate cells on selective medium containing antibiotics.
   • Only transformed cells with antibiotic resistance gene on plasmids will grow.

Conclusion

• Successful transformation results in bacteria expressing the gene of interest and antibiotic resistance.

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