Welcome! to the “Modern Lab” section
description
Transcript of Welcome! to the “Modern Lab” section
![Page 1: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/1.jpg)
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!!!
![Page 2: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/2.jpg)
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
![Page 3: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/3.jpg)
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
![Page 4: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/4.jpg)
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
![Page 5: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/5.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
![Page 6: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/6.jpg)
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)
![Page 7: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/7.jpg)
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.
![Page 8: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/8.jpg)
PCR movie
![Page 9: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/9.jpg)
Day2: LigationInserting your DNA into a plasmid
cellDNA
GFP (protein)
How?
How?
![Page 10: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/10.jpg)
Day2: LigationInserting your DNA into a plasmid
cellDNA
GFP (protein)
The answer is a vector !
![Page 11: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/11.jpg)
Day2: LigationInserting your DNA into a plasmid
Piece of DNA…
…in a circle
vector
![Page 12: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/12.jpg)
Day2: LigationInserting your DNA into a plasmid
vector
GFP DNA
Need to insert GFP DNA into vector DNA
![Page 13: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/13.jpg)
Day2: LigationInserting your DNA into a plasmid
vector
GFP DNA
Restriction Enzyme
cut
cut
![Page 14: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/14.jpg)
Day2: LigationInserting your DNA into a plasmid
G A A T T C
C T T A A G
![Page 15: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/15.jpg)
Day2: LigationInserting your DNA into a plasmid
vector
insert“Ligation”
![Page 16: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/16.jpg)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
cell
Vector + GFP DNA
GFP (protein)
![Page 17: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/17.jpg)
plasmid
42oCelsiusHeat shock
Bacterial chromosome
Bacterium
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
![Page 18: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/18.jpg)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Basic plasmid
Selection marker
Origin of replication
Signal to read DNA
Resistance gene
![Page 19: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/19.jpg)
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!)
![Page 20: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/20.jpg)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Result is a cell with multiple copies of the plasmid carrying your GFP DNA insert
![Page 21: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/21.jpg)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Petri dish with agar
Streak onto agar plate
![Page 22: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/22.jpg)
Day3: TransformationInserting your GFP-plasmid into a bacterial cell
Selection marker
Food source + ampicillan
![Page 23: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/23.jpg)
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
![Page 24: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/24.jpg)
Day4: ExpressionTranscribing DNA RNATranslating RNA green fluorescent protein
Peyer movie
![Page 25: Welcome! to the “Modern Lab” section](https://reader035.fdocuments.in/reader035/viewer/2022062222/56815753550346895dc4f873/html5/thumbnails/25.jpg)
Day5: Fluorescence
cell