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Welcome! to the “Modern Lab” section

Welcome! to the “Modern Lab” section. Graduate students: Francis Raycroft , Aprell Carr, & Aranda Slabbekoorn. Why are you running a modern lab experiment? This is a treat to yourself as a scientist!!!. Cloning a Fluorescent Gene. Detailed lab manual ( Peyer Laboratory Systems, LLC)

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Welcome! to the “Modern Lab” section

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  1. Welcome!to the “Modern Lab” section Graduate students: Francis Raycroft, Aprell Carr, & ArandaSlabbekoorn Why are you running a modern lab experiment? This is a treat to yourself as a scientist!!!

  2. Cloning a Fluorescent Gene • Detailed lab manual (Peyer Laboratory Systems, LLC) • Week-long lab • Work in pairs WHAT YOU WILL DO • PCR, bacterial transformation, protein expression… • Will clone the protein GFP into E.coli cells

  3. Aequoriavictoria jellyfish • Dr. Shimomura in 1961 • Green Fluorescent Protein “GFP” • GFP successfully expressed in E.coliin 1994 http://3.bp.blogspot.com http://www.animalpicturesarchive.com http://www.nature.com

  4. Cloning GFP • Characterized the GFP protein • Sequenced the protein’s DNA • Cloned GFP DNA • Attach it to any other protein in the cell that one wants to study • Whatever you want to study is now visibly fluorescing and can be seen under a microscope • THE MICROSCOPIC WORLD IS OPENED TO OUR EYES! http://www.olympusfluoview.com/applications/images/fpcolorpalettefigure2.jpg

  5. Significance & Application Light Microscopy GFP-proteins using fluorescence microscopy http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE428300FG0010.gif http://www.bioelcomind.de/gallery/MDCK_cell.jpg

  6. Significance & Application 2 Days : GFP labeling the circulatory system 4 Days : GFP labeling proteins in nuerons (central nervous system) www.ucl.ac.uk/.../research/neuroanatomy.php www.exploratorium.edu/imaging_station/gallery...

  7. Day1: Replicate the DNAPolymerase Chain Reaction (PCR) • Takes a piece of DNA and duplicates it over and over • Couple examples of importance: • Forensics: a very tiny DNA sample can be • amplified by PCR. • There is then more DNA to work with • and run different tests on. • Biochemistry: can use cloned DNA to transform • and express protein in large • amounts. • Circumvents having to use live tissue • samples.

  8. PCR movie

  9. Day2: LigationInserting your DNA into a plasmid DNA cell How? How? GFP (protein)

  10. Day2: LigationInserting your DNA into a plasmid DNA cell The answer is a vector ! GFP (protein)

  11. Day2: LigationInserting your DNA into a plasmid Piece of DNA… …in a circle vector

  12. Day2: LigationInserting your DNA into a plasmid GFP DNA Need to insert GFP DNA into vector DNA vector

  13. Day2: LigationInserting your DNA into a plasmid GFP DNA cut cut vector Restriction Enzyme

  14. Day2: LigationInserting your DNA into a plasmid G A A T T C C T T A A G

  15. Day2: LigationInserting your DNA into a plasmid insert “Ligation” vector

  16. Day3: TransformationInserting your GFP-plasmid into a bacterial cell cell Vector + GFP DNA GFP (protein)

  17. Day3: TransformationInserting your GFP-plasmid into a bacterial cell plasmid 42oCelsius Heat shock Bacterium Bacterial chromosome

  18. Day3: TransformationInserting your GFP-plasmid into a bacterial cell Origin of replication Basic plasmid Signal to read DNA Selection marker Resistance gene

  19. Day3: TransformationInserting your GFP-plasmid into a bacterial cell = DNA polymerase Origin of replication • DNA polymerase recognizes the origin of replication • Begins to replicate the plasmid • (plus your inserted GFP sequence!) Basic plasmid Selection marker

  20. Day3: TransformationInserting your GFP-plasmid into a bacterial cell Result is a cell with multiple copies of the plasmid carrying your GFP DNA insert

  21. Day3: TransformationInserting your GFP-plasmid into a bacterial cell Streak onto agar plate Petri dish with agar

  22. Day3: TransformationInserting your GFP-plasmid into a bacterial cell Food source + ampicillan Selection marker

  23. Day3: TransformationInserting your GFP-plasmid into a bacterial cell Food source + ampicillan • Drug in the agar • Antibiotic resistance in the plasmid • All cells without the plasmid die • All cells that took up your GFP-plasmid will survive Selection marker

  24. Day4: ExpressionTranscribing DNA  RNA Translating RNA green fluorescent protein Peyer movie

  25. Day5: Fluorescence cell

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