Observe the given picture carefully. A mixture of DNA with fragments ranging from 100 base pairs to 1800 base pairs were separated by electrophoresis on agarose gel with the following arrangement:
Question: 1
What result will be obtained in staining with ethidium bromide? Explain with reasons.
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Always place the wells near the negative (black) electrode so that DNA can run "Run to Red" (towards the positive red electrode).
Step 1: Understanding the Concept:
Gel electrophoresis separates DNA based on size using an electric field. DNA molecules are negatively charged. Step 2: Detailed Explanation:
1. Reasoning: DNA fragments have a net negative charge due to the phosphate groups in their backbone.
2. Direction of movement: In an electric field, DNA always migrates toward the Anode (positive electrode).
3. Observation: In the provided diagram (28a), the positive electrode (+) is at the top where the wells are located, and the negative electrode (-) is at the bottom.
4. Result: Instead of migrating down through the gel, the DNA will stay in the wells or migrate out of the top of the wells toward the (+) electrode. No separation will occur within the agarose matrix. Step 3: Final Answer:
No bands will appear because the polarity is reversed; DNA will not enter the gel.
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Question: 2
The above setup was modified as shown below and a band with 100 base pairs was obtained at X. What changes were made to the previous design to get a band at 'X'? Why did the band appear at 'X'?
Show Hint
The distance traveled by a DNA fragment is inversely proportional to the log of its molecular weight (number of base pairs).
Step 1: Understanding the Concept:
The agarose gel acts as a sieve. The speed of migration is inversely proportional to the size of the DNA fragment. Step 2: Detailed Explanation:
1. Changes made: The electrodes were swapped so that the wells are now at the negative (-) pole and the bottom of the gel is at the positive (+) pole. This allows the negatively charged DNA to migrate down through the gel.
2. Position X: The position 'X' is the furthest distance from the wells.
3. Reasoning: In gel electrophoresis, smaller fragments experience less resistance from the agarose matrix and move faster than larger fragments.
4. Since 100 bp is the smallest fragment in the mixture (100-1800 bp), it will travel the maximum distance and form the band at the bottom (position X). Step 3: Final Answer:
The polarity was corrected, and the 100 bp fragment traveled furthest because it is the smallest.
