gel electrophoresis 08
TRANSCRIPT
![Page 1: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/1.jpg)
Gel ElectrophoresisGel Electrophoresis
http://biotrek.rdrake.org/img/gallery/33electrophoresis1_training.jpg
![Page 2: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/2.jpg)
http://www.antibiotic.ru/en/mbio/pub/images/p233r2.gif
DefinitionDefinition
Gel electrophoresis is a technique used to separate DNA bands based on size
![Page 3: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/3.jpg)
Quick QuizQuick Quiz
Gel electrophoresis separates molecular products based on:
A. ShapeB. SizeC. ChargeD. Evolutionary similarity
![Page 4: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/4.jpg)
ExamplesExamples
Gel Electrophoresis is used to visualize…
Genomic DNARNAPCR productsPlasmidsRestriction enzyme
digest productshttp://www.steve.gb.com/images/science/agarose.jpg
![Page 5: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/5.jpg)
Non-exampleNon-example
Gel Electrophoresis is not used to…
Separate DNA from the cell or from tissues
![Page 6: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/6.jpg)
AgaroseAgarose
Neutral polysaccharide
Harvested from Rhodophyceae algal cell wallshttp://www.steve.gb.com/images/
![Page 7: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/7.jpg)
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
![Page 8: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/8.jpg)
Quick QuizQuick Quiz
When traveling through an agarose gel, larger molecular products will migrate _________ smaller molecular products.
A. Faster thanB. Slower thanC. At the same rate
![Page 9: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/9.jpg)
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
![Page 10: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/10.jpg)
http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/gelelectrophoresis.html
http://learn.genetics.utah.edu/units/biotech/gel
How Gel Electrophoresis WorksHow Gel Electrophoresis Works
![Page 11: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/11.jpg)
Quick QuizQuick Quiz
As DNA has a net _______ charge, it migrates towards the gel box’s _________.
A. Positive; cathodeB. Positive; anodeC. Negative; cathodeD. Negative; anode
![Page 12: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/12.jpg)
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
![Page 13: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/13.jpg)
Quick QuizQuick Quiz
The size of the molecular products is determined by:
A. The intensity of the bandB. The percentage of agarose gelC. Comparison with a molecular weight “ladder”D. Being familiar with your gel and “eyeballing” it
![Page 14: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/14.jpg)
Gel Electrophoresis Gel Electrophoresis
Prepare agarose gelMelt, 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
![Page 15: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/15.jpg)
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 waterC. Buffer neutralizes the charge on molecular products
prior to electrophoresisD. Buffers can be customized based upon the
electrophoresis protocol
![Page 16: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/16.jpg)
Troubleshooting GuideTroubleshooting Guide
DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer
![Page 17: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/17.jpg)
ENDEND
![Page 18: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/18.jpg)
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/majorsbiology/gelelectrophoresis.html
http://learn.genetics.utah.edu/units/biotech/gel
![Page 19: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/19.jpg)
California State Chemistry Standards California 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…
![Page 20: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/20.jpg)
California State Biology StandardsCalifornia State Biology Standards
Grades 9-12
5d. Students know the general structure and function of DNA
![Page 21: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/21.jpg)
California State California 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
![Page 22: Gel electrophoresis 08](https://reader035.vdocuments.us/reader035/viewer/2022062316/58ee6a121a28ab96438b469f/html5/thumbnails/22.jpg)
National StandardsNational Standards
Grades 6-12
Content Standard A: Science as Inquiry
Content Standard C: Life Science
Content Standard E: Science and Technology