Southern blot: Difference between revisions
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The '''Southern blot''' is a technique to detect the presence of a specific piece of DNA sequence. It is named after Edward M. Southern who developed the technique at Edinburgh University in the 1970 <ref>http://lifesciences.asu.edu/resources/mamajis/southern/southern.html</ref>. | The '''Southern blot''' is a technique to detect the presence of a specific piece of DNA sequence. It is named after Edward M. Southern who developed the technique at Edinburgh University in the 1970 <ref>http://lifesciences.asu.edu/resources/mamajis/southern/southern.html</ref>. | ||
==The Process== | ==The Process== | ||
The DNA in which has to be tested for the specific sequence | The DNA in which has to be tested for the specific sequence is cut into pieces by a [[restriction enzyme]]. | ||
Afterwards [[gel electrophoresis]] is used to separate those pieces by size on an [[agarose]] gel. | Afterwards, [[gel electrophoresis]] is used to separate those pieces by size on an [[agarose]] gel. | ||
[[Alkali]] is used to | [[Alkali]] is used to denature the DNA fragments and the denatured DNA is then transferred to a [[nitrocellulose filter]] or [[nylon membrane]] by blotting. | ||
The filter | The filter is incubated with a single stranded DNA probe, which hybridizes with the specific sequences to which it is targeted. That probe is either radioactively labeled (using 32P) or marked with an enzyme. Excess probe is removed by extensive washing. | ||
If the probe is | If the probe is labeled radioactively it can be detected with X-ray sensitive film, or more recently, fluorescent imagers have been used. If the label is an enzyme, a substrate is added that develops a color through interaction with the enzyme. | ||
<ref>[http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.section.1692 Molecular Cell Biology 4ed by Lodish et al]</ref><ref>http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html</ref>. | <ref>[http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.section.1692 Molecular Cell Biology 4ed by Lodish et al]</ref><ref>http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html</ref>.--[[User:Raymond Shillito|Raymond Shillito]] 21:05, 19 September 2008 (CDT) | ||
Revision as of 20:05, 19 September 2008
The Southern blot is a technique to detect the presence of a specific piece of DNA sequence. It is named after Edward M. Southern who developed the technique at Edinburgh University in the 1970 [1].
The Process
The DNA in which has to be tested for the specific sequence is cut into pieces by a restriction enzyme.
Afterwards, gel electrophoresis is used to separate those pieces by size on an agarose gel.
Alkali is used to denature the DNA fragments and the denatured DNA is then transferred to a nitrocellulose filter or nylon membrane by blotting.
The filter is incubated with a single stranded DNA probe, which hybridizes with the specific sequences to which it is targeted. That probe is either radioactively labeled (using 32P) or marked with an enzyme. Excess probe is removed by extensive washing.
If the probe is labeled radioactively it can be detected with X-ray sensitive film, or more recently, fluorescent imagers have been used. If the label is an enzyme, a substrate is added that develops a color through interaction with the enzyme. [2][3].--Raymond Shillito 21:05, 19 September 2008 (CDT)