JoVE Lab Manual
Biologia
Bacterial Transformation

Bacterial Transformation

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Lab Manual
Biologia
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Lab Manual Biologia
Bacterial Transformation
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January 31, 2019

Learning Objectives

What is transformation?

Transformation is the intake of DNA into a cell from the environment.

Why are bacteria commonly used in the lab for transformation?

Bacteria grow rapidly and can easily take up genetic material from their environment.

What is a plasmid and why is it used routinely for transformation in the lab?

A plasmid is a small loop of DNA that has been developed to facilitate the cloning process. These plasmids can be easily tracked through antibiotic resistance or fluorescent markers and genes of interest can be easily inserted into the plasmid.

How is bacterial transformation used in biotechnology?

Bacterial transformation is a step in the cloning process, which allows us to genetically modify organisms. For example, cloning gives us the ability to produce large quantities of protein such as insulin.

How can we induce bacteria to take up DNA?

By suspending bacterial cells into a solution of calcium chloride and heat-shocking, the cell membranes become porous. This allows the plasmid to enter the cell.

List of Materials

Print Materials List
  • pGreen Plasmid (0.005 µg/µL)
    10 µL
  • E. coli (OP50 strain) (1 tube)
    5 tubes
  • 1M CaCl2
    5 mL
  • LB broth media
    500 mL
  • LB+AMP agar plates (100µg/mL AMP)
    10
  • LB agar plates
    20
  • SOC media
    10 mL
  • Goggles
    5
  • Inoculating loops
    5
  • Laboratory film/ Parafilm
    1 per station
  • 70% Ethanol spray bottle
    1 per station
  • Microfuge tubes (1.5mL)
    1 full beaker/station
  • Pipettes (1- 1000µL)
    1 set/station
  • Pipett tips (tip boxes) (1- 1000µL)
    1 set/station
  • Incubator (Set to 37°C)
    -1 Dependent on the lab size
  • Water bath (Set to 42°C)
    -1 Dependent on the lab size
  • UV Transilluminator
    -1 Dependent on the lab size

Preparação do Laboratório

Print Setup Guide
  1. Bacterial Transformation
    • First, ensure that each student or student group has a filled ice bucket.
    • Then, set up a tube rack for each work station.
    • Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
    • Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
    • Set out two LB agar plates per group and two LB agar ampicillin plates per group.
    • Ensure the students have the appropriate pipettes.
    • And finally, set a water bath to 42 °C and a temperature-controlled shaker to 37 °C.

Tags

JoVE LabLab: 11 Prep