Lecture 6: DNA Cloning and Isolating Genes
After reading through this lecture supplement, you should be able to answer the following questions:1. What are the characteristics of restriction enzymes and how are they useful for DNA cloning?
2. What are cloning vectors? What are the different kinds of cloning vectors? What is the size of DNA insert that can be placed into each of these vectors? How are these vectors used to carry and amplify DNA inserts.
3. What is the basic difference between genomic and cDNA libraries? How are genomic libraries constructed? What is the purpose of having overlapping DNA fragments in genomic libraries? What is 'Reverse Transcriptase?' How is it used to generate cDNA libraries?
4. How might you clone a segment of DNA into a plasmid vector? Use pBR322 as an example of the vector. How would you know that bacteria "took-up" the plasmid construct?
5. How might one isolate a gene for an inherited disorder starting with the protein sequence? What are degenerate primers?
6. What is "reproductive" cloning? What are some of the pitfalls of this method? What is "therapeutic" cloning?
7. How was "Dolly" the sheep cloned? When was she cloned?
8. What is meant by the term, "genetic engineering?"
KIT Corner:
1. What is the underlying theory behind the luciferase reporter assay? Is it a fluorescence assay?
2. What is the underlying theory behing the green fluorescent protein reporter assay? What is the excitation wavelength here? emission wavelength here? why the difference?
3. What is the underlying theory behing the nitric oxide fluorometric assay kit?
posted by Blase Billack, Ph.D. @ 5:24 AM
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