Questions on Southern Blots 1. When hybridizing the probe to the Southern blot,
ID: 280674 • Letter: Q
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Questions on Southern Blots 1. When hybridizing the probe to the Southern blot, the target DNA must be single stranded. When and how do you make the DNA on the nitrocellulose single stranded? 2. You want to determine if the chipmunk genome contains an insulin gene. You isolate chipmunk DNA, digest it with Eco RI, run it on an agarose gel, denature it, and transfer it to nitrocellulose. You have the human insulin gene cloned, so you label it with P, denature it, and hybridize it to the Southern blot. When you develop the Xray film, you see two bands on the Southern hybridization. Why two and not one? Does this tell you if the chipmunk genome has an insulin gene? 3. For a Southern blot, you have run the gel and now have all the DNA fragments separated by size. Why do you have to transfer the DNA to a nitrocellulose filter? 4. What does the salt do when two strands of DNA are hybridized? 5, You are doing a northern blot with a DNA probe that is 200nt long and is 50% GC. In a solution of 2x SSC (IX Standard Saline Citrate = 0.15mM NaCl), what temperature would you use for over night hybridization? (only goto one decimal point)Explanation / Answer
1. Denaturation of DNA is done after the gel electrophoresis to make dsDNA to ssDNA. Because probe can bind with the target sequence of ssDNA only.
The electrophoresis gel is soaked in alkali using NaOH or acid using HCl to denature the dsDNA fragments. Alkali treatment is more preferable.
Composition of denaturation solution: 1.5 M NaCl/0.5 M NaOH. It is stored at room temperature.
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