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Southern Blot Technique

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Which of the below describes the correct order in which procedures are carried out when using the Southern Blot technique?
(a) Cutting the DNA with restriction enzyme, blotting the DNA, electrophoresis of DNA, hybridizing the DNA with probe.
(b) Cutting DNA with restriction enzyme, electrophoresis of DNA, blotting the DNA, hybridizing the DNA with probe.
(c) Blotting the DNA, electrophoresis of DNA, hybridizing the DNA with probe, cutting DNA with restriction enzyme.
(d) Electrophoresis of DNA, hybridizing the DNA with probe, cutting DNA with restriction enzyme, blotting the DNA.
(e) Cutting DNA with restriction enzyme, hybridizing the DNA with probe, electrophoresis of DNA, blotting the DNA.

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Solution Preview

The correct answer is B.

The steps are below:
1. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.

2. The DNA fragments are then electrophoresed on an agarose gel to separate them by size.

3. If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.

4. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a ...

Solution Summary

This solution contains an overview of the southern blot technique.

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