Aashi Arora MCB250_Discussion2_SP24

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University of Illinois, Urbana Champaign *

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250

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Biology

Date

Feb 20, 2024

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docx

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7

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MCB 250 Spring 2024 Discussion Worksheet - 2 Feb. 1-5 1. Write the Henderson-Hasselbach equation. pH = pKA + log([A-]/[HA]) Explain how this equation relates the concentration of the charged and uncharged forms of the side chain of aspartic acid (pKa 4) to pH. The side chain can exist in both charged and uncharged forms be where HA represents the uncharged form and A- represents the charged form. Define pH and pKa. Then, use the equation to determine the fraction of aspartic acid that is charged when: a. the pH = pKa. pH equals pKa, charged and uncharged forms are equal. b. the pH is 1 pH unit below the pKa. pH is 1 unit below pKa so the charged form is 0.1 times the concentration of the uncharged form. c. the pH is 1 pH unit above the pKa. pH is 1 unit above pKa so the charged form is 10 times the concentration of the uncharged form. d. Draw a graph of fraction charged vs. pH for the side chain of Asp. Charged vs pH for the side chain. The graph with show the sigmoidal curve and indicates a transition between the charged and uncharged forms. The midpoint of the transition corresponds to the pKa of aspartic acid. As pH deviates from the pKa, the fraction of the charged form increases or decreases exponentially.
2. The T m of a certain dsDNA is 65°C in a solution buffered at pH 7.2 and containing 0.05 M NaCl. Predict the effect of the following changes on the T m of this DNA. Explain your reasoning in each case. a. Increasing the salt concentration to 0.2 M. Increasing salt concentration will stabilize the DNA duplexes, leading to a higher Tm because higher salt concentrations shield the negatively charged phosphate groups of DNA, reducing repulsion between strands. b. Adding urea to a concentration of 40%. Urea disrupts hydrogen bonding and destabilizes DNA, leading to a lower Tm. c. Doubling the DNA concentration. Increasing DNA concentration tends to increase Tm. Higher DNA concentration provides more potential binding sites for hydrogen bonding making the duplex more stable. d. Changing the pH to 11.0. Higher pH can lead to a decrease in Tm. The increased concentration of hydroxide ions at the higher pH can disrupt hydrogen bonding In DNA, making it less table. e. Adding ethanol to the solution up to 50%. Ethanol can denature DNA and decrease Tm. Ethanol disrupts the hydrogen bonding between DNA strands, leading to decreased stability and a lower Tm. f. Replacing the NaCl with KCl at the same 0.05 M concentration. KCl is expected to have a similar stabilizing effect as NaCl so the Tm is unchanged. Both Na+ and K+ ions stabilize DNA through similar mechanisms. The change in cation type is less likely to significantly affect Tm.
3. Answer the following questions regarding the short nucleic acid shown to the right. a. Is this DNA or RNA? How do you know? DNA due to the presence of thymine instead of uracil b. Mark the 5’ and 3’ ends of the strand. 5’ end is at the top and 3’ end is at the bottom. c. Circle and name the chemical groups that mark these two ends. The Phosphate group is the 5 end and the pentose sugar at the bottom for the 3 end. d. What is the base sequence? G A T e. What is the complementary base sequence? CTA 4. Several DNA samples have been separated by agarose gel electrophoresis and stained with ethidium bromide. In the gel shown at the right, Lane 5 contains standard DNA fragments with the size (in base 400 240 160 80 bps ng/band
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