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Unformatted text preview: Exercise 5 Analysis Answer all questions individually first. Then, meet as a group to compile and sumbit a cohesive answer and one printed report submitted at the beginning of the next laboratory period. Include YOUR Data & Results , sketches, acquired digital images, etc. as directed by your laboratory instructor. Show your work for all calculations and/or print your MS Excel data sheets. When reporting YOUR values, be sure to include proper units where necessary. Adhere to UTs Honor Code and course policies. If you use a secondary resource, cite it! Balance between brevity and completeness. (1) Demonstrate your understanding of agarose gel electrophoresis. (a.) How does agarose gel electrophoresis work to separate different size fragments of DNA? (b.) In what direction does DNA migrate: toward the anode or cathode? Explain your answer. (c.) If one wanted to separate DNA fragments of very large sizes, would one increase or decrease the percentage of the agarose in solution to make the gel? For example, which would be better: a 0.7% or 2% agarose gel to separate kb fragments? Explain your answer. (d.) If you wanted your gel to run faster, would you decrease the ionic strength of the TAE running buffer (make it more dilute) or use more concentrated TAE running buffer (increase the ionic strength)? Explain your answer. (e.) List some other factors that would affect the rate of DNA migration through the gel? (2) Demonstrate your understanding of sample preparation and visualization of DNA. (a.) What are the functions of each of the components of the 6 X DNA Loading dye solution? (i.) Xylene Cyanol (ii.) Bromophenol Blue (iii.) SDS sodium dodecyl (iv.) EDTA (v.) Sucrose (or glycerol) (vi.) Ethidium Bromide (b.) DNA by itself is not visible to the naked eye when it is in solution. How can one visualize DNA fragments in a gel? What precautions must be taken when visualizing DNA as done in this exercise? (3) Prepare a standard curve showing the relationship between known size (in bp) of the molecular weight marker/ fragments (bands of the MW ladder) and the migration distance for each of the bands. (c.) Use data from the agarose gel you ran to prepare a scatter plot in MS Excel. Place the logarithm of each of the DNA marker sizes (in bp ) along the Y-axis of the graph and the migration distances on the X-axis. (d.) Perform a linear regression analysis. (e.) Using the linear regression equation, estimate the approximate size of the DNA bands found in each of the PCR samples. Show your work. (4) One often uses gel electrophoresis to confirm that the segment of DNA was amplified during PCR. Certainly, before any PCR product is used in further applications, it should be analyzed to determine whether: i. any PCR product formed at all....
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- Summer '08