Ex #5-Analysis - BIO206L Exercise 5 Analysis To be...

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BIO206L Exercise 5 Analysis To be completed as a group and turned in at the beginning of your 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. Be sure to include proper units where necessary. Adhere to University’s Honor Code and course policies. 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? The electrophoresis separates ion and molecules dissolved in a buffer solution through the gel with the aid of an electrical field. Because the DNA is negatively charged, we set it near the negative pole and thus it is attracted to the positive pole. The larger molecules cannot travel through the pores as far and thus make bands closer to the negative pole. (b.) What are the functions of each of the components of the “6 X DNA Loading dye” solution? The sucrose or glycerol adds density to the solution, while the dyes color so we can tell the difference in the bands and how far they traveled. SDS and EDTA are used to add charges to help the DNA travel through the pores. (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. For large DNA fragments, one would need to decrease the percentage of the agarose in solution because the less concentrated agarose will make the large DNA run faster and smoother. (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. We would want to decrease the ionic strength of TAE running buffer; however, that would cause the DNA fragments blurry due to less concentrated ions in the solution. If we have less ions concentration, then we would have less electrolytes to conduct the charges, the DNA will run quicker. Also with too much TAE buffer, which contains a lot of concentration of ions, then it would risk DNA to melt. (e.) List some other factors that would affect the rate of DNA migration through the gel? There are a couple of other things that could affect the rate of DNA migration. The concentration of the buffer, percentage of agarose in the gel, the voltage used, and increasing the concentration of the loading dye.
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(2) 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.
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Ex #5-Analysis - BIO206L Exercise 5 Analysis To be...

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