Southern blotting workflow Which description best summarizes the core steps of a Southern blot used to detect specific DNA sequences?

Difficulty: Easy

Correct Answer: Electrophoresis of DNA molecules and then blotting the separated DNA bands followed by incubation

Explanation:


Introduction / Context:
Southern blotting is a classic technique for detecting specific DNA fragments after restriction digestion and gel electrophoresis, using hybridization with a labeled probe.



Given Data / Assumptions:

  • Target nucleic acid is DNA.
  • Fragments are separated on an agarose gel.
  • Transfer to a membrane enables stable probe hybridization.


Concept / Approach:
The method proceeds: digest DNA, electrophorese, depurinate/denature, transfer (capillary or electroblot) to a membrane, fix DNA, hybridize with labeled probe, wash, and detect.



Step-by-Step Solution:
Run DNA on agarose gel to resolve sizes.Denature and transfer DNA to a membrane (nylon/nitrocellulose).Hybridize with a complementary labeled DNA/RNA probe.Wash and visualize by autoradiography or chemiluminescence.



Verification / Alternative check:
Controls with known positive and negative fragments confirm specificity and size.



Why Other Options Are Wrong:
Options involving RNA describe Northern blotting; skipping transfer prevents durable detection; proteins correspond to Western blots.



Common Pitfalls:
Poor transfer efficiency; nonstringent washes causing background; probe mishybridization.



Final Answer:
Electrophoresis of DNA molecules and then blotting the separated DNA bands followed by incubation.

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