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Gel Electrophoresis

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Presentation on theme: "Gel Electrophoresis"— Presentation transcript:

1 Gel Electrophoresis

2 Definition Gel electrophoresis is a technique used to separate DNA bands based on size

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

4 Examples Gel Electrophoresis is used to visualize…  Genomic DNA  RNA  PCR products  Plasmids  Restriction enzyme digest products

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

6 Agarose Neutral polysaccharide Harvested from Rhodophyceae algal cell walls

7 Agarose 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

8 Quick 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? 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

10 gelelectrophoresis.html How Gel Electrophoresis Works

11 Quick 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 DNA Ethidium Bromide binds to DNA Fluorescence under UV light makes bands visible

13 Quick 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 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 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 Guide DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer

17 END

18 Resources Gel Electrophoresis Handout DNA Science: A First Course in Recombinant DNA Technology, by David A. Micklos and Greg A. Freyer majorsbiology/gelelectrophoresis.html majorsbiology/gelelectrophoresis.html

19 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…

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

21 California State Investigation and Experimentation Standards 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 Standards Grades 6-12 Content Standard A: Science as Inquiry Content Standard C: Life Science Content Standard E: Science and Technology

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