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Student Guide Tab C pages C1 – C13 Teacher Guide Tab C pages C1 – C15

Abridged Genetic Engineering Sequence. Ch. 2A: How Do You Begin to Clone a Gene? Lab 2A Preparing to Verify the RFP Gene: Digesting the pARA -R Plasmid. Student Guide Tab C pages C1 – C13 Teacher Guide Tab C pages C1 – C15. Learning goals. Describe the characteristics of plasmids

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Student Guide Tab C pages C1 – C13 Teacher Guide Tab C pages C1 – C15

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  1. Abridged Genetic Engineering Sequence Ch. 2A: How Do You Begin to Clone a Gene? Lab 2A Preparing to Verify the RFP Gene: Digesting the pARA-R Plasmid Student Guide Tab C pages C1 – C13 Teacher Guide Tab C pages C1 – C15

  2. Learning goals • Describe the characteristics of plasmids • Explain how plasmids are used in cloning a gene • Describe the function of restriction enzymes • Explain how to use restriction enzymes to create a recombinant plasmid

  3. Key Ideas • Plasmids - ideal vectors for genetic engineering • replicate in the bacteria cell • gene promoter • antibiotic resistance as a selectable marker, • can be transferred into bacteria by conjugation. • Restriction enzymes – key to the creation of a recombinant plasmid. • cut DNA at specific sequences • sticky ends allow strands to join

  4. What’s new? • New pARA-R plasmid

  5. What’s new? 1. Cut the plasmid and the human DNA with the appropriate restriction enzyme 2. Insert the insulin gene into the plasmid DNA 3. Determine which antibiotic you would use to identify bacteria that have taken in the plasmid • Clone That Gene activity

  6. Tips • Reagents should be stored in a freezer until you are ready to prepare them for students. Allow to defrost for 15 minutes before using. • The reagents can be aliquoted up to several days before the lab, then store in the freezer/refrigerator • Vortex and spin enzyme mix and 2.5x RB before aliquoting • Show the video – bio11_vid_smallvolume_450 • Show video – bio11_vid_genengdna video

  7. Tips Multiple pans allow tubes from different classes to stay separated Low-tech water bath Calibrate and mark the controller Bottom of tubes in the water “Floatie” marked with team number. Each period has a different color. “Smart and Final” and “Thrift Store” are super resources!

  8. Restriction digest of pARA-R Biotech Experience Recombinant plasmid of interest BamH I BamH I pARA-R 5,302 bp PBAD-rfp 806 bp Hind III Hind III

  9. Restriction analysis of pARA-R Biotech Experience Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4,496 bp Hind III BamH I 806 bp

  10. Go to Student Guide page C9 and complete Lab 2A

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