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About This Simulation
Dive into recombinant DNA technology with cell division, transcription and translation. Includes concepts in restriction enzymes, cloning and reporter genes.
Learning Objectives
- Understand molecular cloning techniques: DNA extraction and preparation, ligation, transformation, plate streaking and antibiotic selection
- Understand inducible gene expression regulation
- Understand the use of GFP as a reporter gene
- Understand DNA damage and DNA repair system
About This Simulation
Lab Techniques
- Transformation
- Colony screening
- DNA extraction
- Cloning
Related Standards
- Early Stage Bachelors Level
- EHEA First Cycle
- FHEQ 6
- Intermediate Stage Bachelors Level
- Late Stage Bachelors Level
- US College Year 1
- US College Year 2
- US College Year 3
- US College Year 4
- No direct alignment
- No direct alignment
- No direct alignment
Learn More About This Simulation
Molecular cloning is one of the techniques that has laid the foundation for modern biotechnology. The technique was first used in the 1980′s and allowed the insertion of an insulin gene derived from humans to be inserted into yeast and coli bacteria. This allowed the microbes to produce insulin, which is the primary medication in diabetes treatment. Since then, molecular cloning and genetic engineering has become one of the most fundamental techniques ranging from pharmaceutical production, bioethanol production along with medical to basic research. In the Molecular Cloning lab, you will learn how to assemble an expression vector containing RAD52 and GFP. The aim is to control the expression level of RAD52 with Doxycyline and to monitor the expression level by observing the GFP signal.
Vector assembly
In the first part of the Molecular Cloning lab, you will learn how to extract DNA from yeast cells and restrict enzyme isolation in DNA from another vector. First, you will prepare the extracted DNA and measure the concentration, and then, you will assemble a vector containing a gene of interest (RAD52) and GFP using the correct ligase, buffer and temperature of incubation.
Transformation
The assembled vector will be transformed into yeast cells using electroporation. RAD52 gene expression is regulated by a gene regulator. When Doxycyline is added to the media, RAD52 gene will be silenced. GFP is used as a reporter gene to RAD52, cells with active RAD52 will also express GFP and cells with silenced RAD52 will not express GFP. The GFP signal is monitored by exposing the cells to blue light.
DNA damage and repair system
RAD52 is hypothesized to be an important player in DNA repair. You will perform an experiment comparing the result of induced DNA damage through UV radiation in cells expressing RAD52 and cells with silenced RAD52. If RAD52 is important in performing DNA repair, cells with silenced RAD52 will not survive the UV radiation treatment. All in all, the Molecular Cloning lab will give you an overview of the molecular cloning techniques and the reporter gene, and you will learn all about DNA damage and DNA repair system.
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North Dakota State College of Science
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Lecturer in Human Physiology
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San José State University
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FAQs
Find answers to frequently asked questions.
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