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Welcome!to the “Modern Lab”
sectionGraduate 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)
Week-long lab
Work in pairs
WHAT YOU WILL DO
PCR, bacterial transformation, protein expression…
Will clone the protein GFP into E.coli cells
http://www.nature.comhttp://www.animalpicturesarchive.com
http://3.bp.blogspot.com
Aequoria victoria jellyfish
•Dr. Shimomura in 1961•Green Fluorescent Protein “GFP”•GFP successfully expressed in E.coli in 1994
Cloning GFPCharacterized the GFP protein
Sequenced the protein’s DNA
Cloned GFP DNA Attach it to any other protein in the cell that one
wants to studyWhatever you want to study is now visibly
fluorescing and can be seen under a microscopeTHE MICROSCOPIC WORLD IS OPENED TO OUR
EYES!
http://www.olympusfluoview.com/applications/images/fpcolorpalettefigure2.jpg
http://content.answers.com/main/content/img/McGrawHill/Encyclopedia/images/CE428300FG0010.gif http://www.bioelcomind.de/gallery/MDCK_cell.jpg
Significance & Application
Light Microscopy GFP-proteins using fluorescence microscopy
Significance & Application
www.ucl.ac.uk/.../research/neuroanatomy.php
www.exploratorium.edu/imaging_station/gallery...
2 Days : GFP labeling the circulatory system
4 Days : GFP labeling proteins in nuerons (central nervous system)
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.
Day2: LigationInserting your DNA into a plasmid
cellDNA
GFP (protein)
How?
How?
Day2: LigationInserting your DNA into a plasmid
cellDNA
GFP (protein)
The answer is a vector !
Day2: LigationInserting your DNA into a plasmid
Piece of DNA…
…in a circle
vector
Day2: LigationInserting your DNA into a plasmid
vector
GFP DNA
Need to insert GFP DNA into vector DNA
Day2: LigationInserting your DNA into a plasmid
vector
GFP DNA
Restriction Enzyme
cut
cut
Day2: LigationInserting your DNA into a plasmid
G A A T T C
C T T A A G
Day2: LigationInserting your DNA into a plasmid
vector
insert“Ligation”
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
cell
Vector + GFP DNA
GFP (protein)
plasmid
42oCelsiusHeat shock
Bacterial chromosome
Bacterium
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Basic plasmid
Selection marker
Origin of replication
Signal to read DNA
Resistance gene
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Basic plasmid
Selection marker
Origin of replication
= DNA polymerase
•DNA polymerase recognizes the origin of replication
•Begins to replicate the plasmid (plus your inserted GFP sequence!)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Result is a cell with multiple copies of the plasmid carrying your GFP DNA insert
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Petri dish with agar
Streak onto agar plate
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Selection marker
Food source + ampicillan
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Selection marker
•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
Food source + ampicillan
Day4: ExpressionTranscribing DNA RNATranslating RNA green fluorescent protein
Peyer movie
Day5: Fluorescence
cell
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