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Verify recombination by electrophoresis. Digest of rfp gene.

Verify recombination by electrophoresis. Digest of rfp gene. Transform bacteria with recombinant plasmid. Recombination (ligation) of plasmid and rfp gene. Induce expression of rfp gene . Observe bacteria . Digest of plasmid. Journal 12.02.11.

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Verify recombination by electrophoresis. Digest of rfp gene.

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  1. Verify recombination by electrophoresis. • Digest of rfp gene. • Transform bacteria with recombinant plasmid. • Recombination (ligation) of plasmid and rfp gene. • Induce expression of rfp gene. • Observe bacteria. • Digest of plasmid

  2. Journal 12.02.11 • Copy the steps on the other slide in the correct order. • Flip the pages in the book: which steps correspond to labs 2a, 4a, 5a? • At which step is there a process of gene expression? • (Can be answered in 3 columns)

  3. DO: Lab 2a – Restriction digest • 1. Mix reagents, as described in page 2a.3 • Reaction buffer mix • Plasmid • Water (to one test tube) • Add enzyme: from teacher. • 2. Place at 37oC for at least 1 hour.

  4. PreLab 2a – Q & A • Animation: Plasmid digestion • Group papers: • Lab 1 – Conclusions page 1.6 • Lab 2a – Conclusions page 2a.4 • Pour Gel (not in 2013)

  5. http://Plasmid Recombination Gene Cloning Animation

  6. pARA-R construct Recombinant plasmid of interest pARA-R 4720 bp BamH I rfp 702bp Hind III

  7. rfp – Gene for Red Fluorescent Protein, originally from the Sea Anemone Discosoma sp.

  8. To presenting students: The following slide is from the alternative lab sequence, where students also performed the ligation step. The two plasmids: One served as the ‘source’ of the rfp gene, and one as the ‘vector’ which would turn into our pARA-R.

  9. Restriction analysis of pKAN-R and pARA Bruce Wallace pKAN-R BamHI 5,512 bp pARA PBAD-rfp 806 bp 4,872 bp BamHI HindIII 376 bp HindIII

  10. Restriction analysis of pKAN-R and pARA Bruce Wallace Restriction fragments after digest with Hind III and BamH I BamH I Hind III 4,706 bp BamH I Hind III 4,496 bp Hind III BamH I 806 bp Hind III BamH I 376 bp

  11. BamH I sticky end Hind III sticky end Hind III sticky end BamH I sticky end Engineering the Plasmid: ligation of rfp gene into p-ARA

  12. Restriction digest of pARA-R Recombinant plasmid of interest pARA-R 4720 bp rfp 702bp BamH I Hind III

  13. To presenting students: • Refer to Lab3 (background and conclusions) in the student guide for more information. • Key points: • Why 70oC incubation (Denaturation – what does this mean?) • What does ligation have to do with ‘recombinant DNA’?

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