Lucci_Prelab11_Monday2pm

Agarose gel common type of stabilizing medium used

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Agarose gel: Common type of stabilizing medium used for the electrophoretic separation of nucleic acids. Base pairs (bp) Unit for length when measuring DNA, frequently used for smaller fragments Kilobase pairs (kb) Unit for length when measuring DNA, frequently used for larger fragments (1000 base pairs = 1 kilobase pair)
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9. Describe the movement (mobility) of DNA based on size through the agarose gel during electrophoresis. The smaller the DNA fragment, the more rapidly it moves during electrophoresis. 10. Analyze Fig. 11.3 and answer the following: a) How would you title this graph? DNA Fragment Length vs. Distance Migrated b) What is the independent variable? Fragment Length c) What is the dependent variable? Distance Migrated d) Describe how the size of the unknown fragment is determined. A table with standard sizes of kilobase pairs is given, from which a calibration line can be plotted and used to estimate the length of the unknown fragment. 11. List the three dyes in the dye mixture used to follow the progress of the separation and the size of DNA they would co-migrate with. Xylene Cyanol (blue-green) – 2800bp Bromphenol Blue (purple-blue) – 250bp Orange-G (orange) – 50 12. What percentage agarose gel is used in this exercise? When or why would higher or lower concentrations be used? 1.2% (200-10000bp) Higher concentrations (ex. 3%) are used for separation of small DNA molecules (100- 1000bp) while low concentrations (ex. 0.6%) are used for large molecules (10000- 100000bp). 13. How are the wells in agarose gel formed? They are preformed by the casting trays that the gel is poured into after mixing the dry polymer in boiling buffer and set to cool at room temperature.
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