Topic 2.4 Molecular Cloning
In this topic you’ll learn about consructing Recombinant DNA, Restriction Enzyme Digestion, DNA Ligation, Bacterial Transformation, and DNA & Gene Libraries. You will start by clicking on the next button at the bottom of the page to being reviewing the basics elements of laboratory safety.
Upon completion of this module topic, you will:
1. be able to identify compatible restriction sites for cloning.
2. be able to conduct a restriction digestion to prepare DNA fragments and vectors for the construction of recombinant DNA.
3. be able to recombine a digested DNA fragment and plasmid vector in a ligation reaction to make recombinant plasmid DNA carrying a gene of interest.
4. be able to perform a bacterial transformation reaction to transform competent bacterial cells with recombinant plasmid DNA.
5. be able to make selective media plates for plating and growing transformed bacterial cells (antibiotic selective or X-gal).
6. be able to plate transformed bacterial cells on selective plates and identifyresistant colonies.
7. be able to screen resistant colonies for the gene of interest by amplifying individual bacterial colony DNA through Restriction Enzyme Digestion.
8. be able to make an agarose gel, load the gel with your PCR reaction samples, and conduct agarose gel electrophoresis of samples.
9. be able to isolate bacterial colonies carrying the plasmid by analyzing the agarose gel and determine samples containing the gene of interest.
10. be able to make sterilized selective liquid media for growing up one of the bacterial colonies containing the gene of interest to produce large quantities of the desired plasmid DNA.
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