SOUTHERN BLOTTING TECHNIQUE!
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.