1. 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!!!

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. Aequoria victoria jellyfish
Dr. Shimomura in 1961
Green Fluorescent Protein “GFP”
GFP successfully expressed in E.coli in 1994

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!

5. Significance & Application
Light Microscopy
GFP-proteins using fluorescence microscopy

6. Significance & Application
2 Days : GFP labeling the circulatory system
4 Days : GFP labeling proteins in nuerons (central nervous system)

8. Day1: Replicate the DNA Polymerase 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.

10. Day2: Ligation Inserting your DNA into a plasmid
cell
How?
How?
GFP (protein)

11. Day2: Ligation Inserting your DNA into a plasmid
cell
The answer is a vector !
GFP (protein)

12. Day2: Ligation Inserting your DNA into a plasmid
Piece of DNA…
…in a circle
vector

13. Day2: Ligation Inserting your DNA into a plasmid
GFP DNA
Need to insert GFP DNA into vector DNA
vector

14. Day2: Ligation Inserting your DNA into a plasmid
GFP DNA
cut
cut
vector
Restriction Enzyme

15. Day2: Ligation Inserting your DNA into a plasmid
G A A T T C
C T T A A G

16. Day2: Ligation Inserting your DNA into a plasmid
vector

17. Day3: Transformation Inserting your GFP-plasmid into a bacterial cell
Vector + GFP DNA
GFP (protein)

18. Day3: Transformation Inserting your GFP-plasmid into a bacterial cell
42oCelsius
Heat shock
Bacterium
Bacterial
chromosome

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

20. Day3: Transformation Inserting 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

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

22. Day3: Transformation Inserting your GFP-plasmid into a bacterial cell
Streak onto agar plate
Petri dish with agar

23. Day3: Transformation Inserting your GFP-plasmid into a bacterial cell
Food source + ampicillan
Selection marker

24. Day3: Transformation Inserting 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

25. Day4: Expression Transcribing DNA  RNA
Translating RNA  green fluorescent protein
Peyer movie

26. Day5: Fluorescence
cell