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.
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 ...
This solution contains an overview of the southern blot technique.
Gene Cloning, Manipulation, & Applications
1. What is meant by the term "clone"?
Why is it important to obtain large quantities of a particular DNA sequence?
List the steps you would take to obtain a clone of a DNA sequence.
2. For each of the following, give a brief description:
recombinant DNA molecule
3. What is the process of transformation?
What is meant by competent cells?
What would you use a selection system for?
4. What information could you obtain from a southern blot?
What information could you obtain from a northern blot?
Describe what a genomic library is.
5. You are a forensic scientist and you perform a southern analysis to try and link a possible suspect to the scene of a crime. Draw the results of such a southern analysis and decide if the suspect was present or not.
Describe a possible application for making a transgenic animal or plant.