SOUTHERN BLOTTING TECHNIQUE!

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Southern Blotting Technique: It is named after its originator. Let us deal what exactly it says. Blotting is a process of transferring the fragments in a gel to a membrane made of Nitrocellulose or Nylon. Procedure: 1. The DNA is cut with a suitable Restriction Endonucleases and run on a gel. 2. The DNA is denatured by treatment with an alkali (NaOH). 3. Southern Blot is then performed by keeping the gel on a stack of filter papers placed in a buffer solution. On the top of the gel, a Nitrocellulose paper is placed above which few more dry filter papers are present. 4. The buffer traverse the filter papers through the gel membrane into top layers. The flow carries the DNA fragments on the gel to the Nitrocellulose where they are entrapped. 5. The membrane with DNA fixed is incubated with labelled probe. 6. Stringent washings are done to remove non-hybridized probe. 7. Autoradiography and subsequent development of X-ray film detects the bands with a bound probe. Applications: 1. This technique is used to seperate the gel fractionated DNA fragments. 2. Identify the discrete restriction fragments arising from a cloned chromosomal segment. 3. Molecular configurations of DNA can be known. THIS BLOG IS FREE OF PLAGIARISM. Reference: An Introduction to Recombinant DNA Technology, Chapter #2, Principles of Gene Manipulation - An Introduction to Genetic Engineering, RW Old, S B Primrose 5th Edition, BlackWell Science Unit, Page Nos: 43-45.
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Sirisha Pingali's picture
Author: Sirisha Pingali

Comments

Siva Mavuduru's picture

Hi Dear why nylon or nitrocellulose is used mostly but not the other gels. what are the other gels used?
Sirisha Pingali's picture

thank you for the valuable comment. Nitrocellulose membrane is widely used in the blotting techniques. You can attribute this to the following reasons: 1. High quality which is ideal for blotting proteins and nucleic acids 2. Compatible with commonly used methods like staining, autoradiography etc 3. Provides high sensitivity with low background 4. Available easily in sandwich forms or filter papers for practical purpose.

Sirisha Pingali

http://www.pharmainfo.net/sirisha

Viswanadha Institute of Pharmaceutical Sciences.

www.vnips.edu.in

SS Md Shafi's picture

dear sirisha... nice blog.... is there any disadvantages in this method ?

shafi ..

Sirisha Pingali's picture

hello shafi, thank you for the valuable comment. 1. Major disadvantage is any blotting require prior electrophoretic seperation. 2. As we can judge with any other process, it is complex and cumbersome. 3. A single gene or RNA/DNA can be studied at a time 4. No information regarding gene interaction or gene regulation is known. Ref: http://www.molecular-plant-biotechnology.info/blotting-techniques/disadv... accessed as on 14th August 2011.

Sirisha Pingali

http://www.pharmainfo.net/sirisha

Viswanadha Institute of Pharmaceutical Sciences.

www.vnips.edu.in

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