Gel electrophoresis in prepration of plasmid DNA

1. Gel ElectrophoresisGel Electrophoresis
http://biotrek.rdrake.org/img/gallery/33electrophoresis1_training.jpg

2. http://www.antibiotic.ru/en/mbio/pub/images/p233r2.gif
DefinitionDefinition
 Gel electrophoresis
is a technique used
to separate DNA
bands based on size

3. Quick QuizQuick Quiz
Gel electrophoresis separates molecular
products based on:
A. Shape
B. Size
C. Charge
D. Evolutionary similarity

4. ExamplesExamples
 Gel Electrophoresis
is used to
visualize…
Genomic DNA
RNA
PCR products
Plasmids
Restriction enzyme
digest products
http://www.steve.gb.com/images/science/agarose.jpg

5. Non-exampleNon-example
 Gel Electrophoresis is not used to…
Separate DNA from the cell or from tissues

6. AgaroseAgarose
 Neutral
polysaccharide
 Harvested from
Rhodophyceae algal
cell wallshttp://www.steve.gb.com/images/

7. AgaroseAgarose
 Alternating sugar units
form linear chains
 Cross-linking chains via
hydrogen bonds forms
porous matrix
 Heat breaks matrix
apart
 Cooling forms a matrix
with an “average” pore
size
http://www.bioscience-beads.com/images

8. Quick QuizQuick Quiz
When traveling through an agarose gel, larger
molecular products will migrate _________
smaller molecular products.
A. Faster than
B. Slower than
C. At the same rate

9. Why Run a Gel?Why Run a Gel?
 Molecular weight
markers are DNA
fragments of known
size
 Comparison of sample
bands to markers
allows…
 Visible confirmation of
desired product
 Quantification of sample
DNA
http://www.biomedcentral.com/content/figures/1471-2180-5-63-2.jpg

10. http://www.sumanasinc.
com/webcontent/anisamples/majorsbiology/gelelectrophoresis.html
http://learn.genetics.utah.edu/units/biotech/gel
How Gel Electrophoresis WorksHow Gel Electrophoresis Works

11. Quick QuizQuick Quiz
As DNA has a net _______ charge, it migrates
towards the gel box’s _________.
A. Positive; cathode
B. Positive; anode
C. Negative; cathode
D. Negative; anode

12. Visualizing DNAVisualizing DNA
 Ethidium Bromide
binds to DNA
 Fluorescence under
UV light makes
bands visible
http://www.lanl.gov/news/albums/bioscience/AnthraxUVplate.sized.jpg

13. Quick QuizQuick Quiz
The size of the molecular products is
determined by:
A. The intensity of the band
B. The percentage of agarose gel
C. Comparison with a molecular weight “ladder”
D. Being familiar with your gel and “eyeballing” it

14. Gel ElectrophoresisGel Electrophoresis
Prepare agarose gel
Melt, cool and add Ethidium Bromide. Mix thoroughly.
Add running buffer, load samples and marker
Run gel at constant voltage until band separation occurs
Pour into casting tray with comb and allow to solidify
View DNA on UV light box and document results

15. Quick QuizQuick Quiz
Buffer is used instead of water when making
and running gels because:
A. Buffer enhances the transmission of electric
currents in water
B. Buffer is more homogeneous than water
C. Buffer neutralizes the charge on molecular products
prior to electrophoresis
D. Buffers can be customized based upon the
electrophoresis protocol

16. Troubleshooting GuideTroubleshooting Guide
DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer

17. ENDEND

18. ResourcesResources
 Gel Electrophoresis Handout
 DNA Science: A First Course in Recombinant DNA
Technology, by David A. Micklos and Greg A. Freyer
 http://www.sumanasinc.com/webcontent/anisamples/majors
 http://learn.genetics.utah.edu/units/biotech/gel

19. California State Chemistry StandardsCalifornia State Chemistry Standards
Grades 9-12
2. Properties of matter result from the ability of atoms to form
bonds…
6. Solutions are homogenous mixtures of two or more
substances
10b. Students know the bonding characteristics of carbon…

20. California State Biology StandardsCalifornia State Biology Standards
Grades 9-12
5d. Students know the general structure and function of DNA

21. California StateCalifornia State
Investigation and ExperimentationInvestigation and Experimentation
StandardsStandards
Grade 7
a. Select and use appropriate tools and technology to perform tests,
collect data, analyze relationships, and display data
Grades 9-12
c. Identify possible reasons for inconsistent results, such as
sources of error or uncontrolled conditions
d. Formulate explanations by using logic and evidence
k. Recognize the cumulative nature of scientific evidence
l. Analyze situations and solve problems that require combining
and applying concepts from more than one area of science

22. National StandardsNational Standards
Grades 6-12
Content Standard A: Science as Inquiry
Content Standard C: Life Science
Content Standard E: Science and Technology