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BIO311: Molecular Biology

Unit 10: Techniques   The classical core technique in molecular biology is cloning.  The cloned DNA is usually recombinant DNA, wherein a plasmid DNA and the gene of interest are pieced together.  Techniques to amplify, analyze, and mutate DNA are also discussed here.  Please take the BIO403: Biotechnology course if you would like to learn more about the array of techniques employed in molecular biology.   

Unit 10 Time Advisory
This unit should take you approximately 20 hours to complete.

☐    Subunit 10.1: 5.5 hours

☐    Subunit 10.2: 2.0 hours

☐    Subunit 10.3: 12.5 hours

☐    Subunit 10.3.1: 3.0 hours

☐    Subunit 10.3.2: 8.0 hours

☐    Subunit 10.3.3: 1.5 hours

Unit10 Learning Outcomes
Upon successful completion of this unit, students will be able to: - Describe techniques to analyze, amplify, and modify DNA. - Outline a strategy for making recombinant DNA. - Describe techniques to purify and analyze proteins. - Compare and contrast the uses of model organisms; discuss the uses of model organisms in specific molecular biology applications. 

10.1 Nucleic-Acid Techniques   10.1.1 Gel Electrophoresis   - Reading: Scitable's "Gel Electrophoresis Can be Used to Separate DNA Molecules on the Basis of Their Size and Electrical Charge" Link:  Scitable's "Gel Electrophoresis Can be Used to Separate DNA Molecules on the Basis of Their Size and Electrical Charge"  (HTML)
 
Instructions:  Please study the schema of gelelectrophoresis.
 
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  • Reading: Davidson College's "Capillary Electrophoresis" Link:  Davidson College's "Capillary Electrophoresis" (HTML)
     
    Instructions:  Please study this page.
     
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10.1.2 Restriction Endonucleases and Cloning   - Reading: John W. Kimball’s “Restriction Enzymes" Link:  John W. Kimball’s “Restriction Enzymes" (HTML)
 
Instructions: Please study this page.  Restriction endonucleases are molecular scissors that cut dsDNA at specific recognition sequences.
 
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10.1.3 Polymerase Chain Reaction (PCR)   - Reading: John W. Kimball’s “The Polymerase Chain Reaction (PCR): Cloning DNA in the Test Tube" Link:  John W. Kimball’s “The Polymerase Chain Reaction (PCR): Cloning DNA in the Test Tube" (HTML)
 
Instructions: Please study this page.  Please recall that E. coli is a prokaryote, thus it lacks membrane-bound subcellular organelles.
 
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  • Web Media: CSHL: DNA Learning Center’s "Finding DNA to Copy, Kary Mullis" Link: CSHL: DNA Learning Center’s "Finding DNA to Copy, Kary Mullis" (Adobe Video)
     
    Instructions:  Please watch this video (1min).
     
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10.1.4 DNA Sequencing   - Reading: John W. Kimball’s “DNA Sequencing" Links:  John W. Kimball’s “DNA Sequencing"(HTML)
 
Instructions:  Please study this page.
 
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  • Reading: NCBI Bookshelf: Koonin and Galperin’s "Principles and Methods of Sequence Analysis" Links:  NCBI Bookshelf: Koonin and Galperin’s "Principles and Methods of Sequence Analysis" (HTML)
     
    Instructions:  Please study this page.
     
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  • Reading: Scitable: Jill U. Adams’s “Simple Viral and Bacterial Genomes" Links:  Scitable: Jill U. Adams’s “Simple Viral and Bacterial Genomes" (HTML)
     
    Instructions:  Please study this page.
     
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10.2 Protein Techniques   10.2.1 Protein Purification   - Reading: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" Link: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" (HTML)
 
Instructions:  Please study the "Proteins Can Be Separated by Chromatography" and "Affinity Chromatography Exploits Specific Binding Sites on Proteins" sections on this page. 
 
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10.2.2 Sodium Dodecyl Sulfate- Polyacrylamide Gel (SDS-PAGE)   - Reading: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" Link: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" (HTML)
 
Instructions:  Please study "The Size and Subunit Composition of a Protein Can Be Determined by SDS Polyacrylamide-Gel Electrophoresis" and "More Than 1000 Proteins Can Be Resolved on a Single Gel by Two-dimensional Polyacrylamide-Gel Electrophoresis" sections on this page. 
 
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10.2.3 Protein Sequencing with Mass Spectroscopy   - Reading: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" Link: NCBI Bookshelf: Alberts et al.'s "Fractionation of Cells" (HTML)
 
Instructions:  Please study the "Selective Cleavage of a Protein Generates a Distinctive Set of Peptide Fragments" and "Mass Spectrometry Can Be Used to Sequence Peptide Fragments and Identify Proteins" sections on this page. 
 
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10.3 Recombinant DNA Technology   10.3.1 Bacterial Plasmid Vectors   - Reading: California State University's "Bacterial Plasmids" Link: California State University's "Bacterial Plasmids" (HTML)
 
Instructions:  Please study this page.
 
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  • Reading: NCBI Bookshelf: Lodish et al.'s "DNA Cloning with Plasmid Vectors" Link:  NCBI Bookshelf: Lodish et al.'s "DNA Cloning with Plasmid Vectors" (HTML)
     
    Instructions: Please study this page.
     
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10.3.2 Cloning   - Reading: NCBI BOOKshelf: Griffiths et al's "Making Recombinant DNA"  Link:  NCBI BOOKshelf: Griffiths et al's "Making Recombinant DNA" (HTML)
 
Instructions: Please study this page.
 
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  • Reading: John W. Kimball’s “Recombinant DNA and Gene Cloning" Link:  John W. Kimball’s “Recombinant DNA and Gene Cloning" (HTML)
     
    Instructions: Please study this page.
     
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  • Web Media: YouTube: MIT: Eric Lander's "Lec 15 | MIT 7.012 Introduction to Biology, Fall 2004" Link: YouTube: MIT: Eric Lander's "Lec 15 | MIT 7.012 Introduction to Biology, Fall 2004"(YouTube)
     
    Instructions: Please watch this video (51 min).
     
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  • Reading: YouTube: MIT: Eric Lander's "Lec 16 | MIT 7.012 Introduction to Biology, Fall 2004" Link: YouTube: MIT: Eric Lander's "Lec 16 | MIT 7.012 Introduction to Biology, Fall 2004"(YouTube)
     
    Instructions: Please watch this video (51 min).
     
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  • Web Media: YouTube: MIT: Eric Lander's "Lec 17 | MIT 7.012 Introduction to Biology, Fall 2004" Link: YouTube: MIT: Eric Lander's "Lec 17 | MIT 7.012 Introduction to Biology, Fall 2004"(YouTube)
     
    Instructions: Please watch this video (51 min).
     
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  • Web Media: YouTube: MIT: Eric Lander's "Lec 18 | MIT 7.012 Introduction to Biology, Fall 2004" Link: YouTube: MIT: Eric Lander's "Lec 18 | MIT 7.012 Introduction to Biology, Fall 2004"(YouTube)
     
    Instructions: Please watch this video (51 min).
     
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  • Assessment: University of Arizona: The Biology Project: "Recombinant DNA Technology Problem Set" Link:  University of Arizona: The Biology Project: "Recombinant DNA Technology Problem Set" (HTML)
     
    Instruction: Please complete this problem set.  There are 11 problems in the set, and each problem is linked to a brief tutorial page.  After answering a question, please click on and study each tutorial page as well.
     
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10.3.4 Site-Directed Mutagenesis   - Reading: Nobelprize.org's "Site-Directed Mutagenesis Reprograms DNA" Links:  Nobelprize.org's "Site-Directed Mutagenesis Reprograms DNA" (HTML)
 
Instructions:  Please study this page.
 
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  • Reading: California State University: Stan Metzenberg's "Site-Directed Mutagenesis Reprograms DNA" Links: California State University:  Stan Metzenberg's "Site-Directed Mutagenesis Reprograms DNA" (HTML)
     
    Instructions:  Please study this page.
     
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