Southern blotting: Difference between revisions
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Southern blotting is | Southern blotting, named after its inventor [[Edwin Southern|Edwin Southern]], is a technique that is used to detect specific [[DNA|DNA]] sequences using gel-transfer hybridization. | ||
Firstly you have to cut the DNA into smaller strands, this is done using a [[restriction enzyme|restriction enzyme]]. Then you need to perform a [[gel electrophoresis|gel electrophoresis]] on [[agrose gel|agrose gel]], using the fragments obtained. The smallest DNA fragments travel the furthest, as they are able to move more easily between the gaps in the agrose gel. Once the gel electrophoresis is complete place a [[Nitrocellulous|nitrocellulous]] sheet over the gel, so the DNA fragments are transfered to the sheet. Wash the sheet in an alkaline solution, for example [[NaOH|NaOH]], this is to ensure that the double stranded DNA is separtaed before hybridization. Place the sheet in a sealed plastic bag thats contains a salt buffered solution and DNA [[nucleotides|nucleotides]] and leave in conditions that favour hybridization for a long period of time. Ensure the DNA nucleotides are labeled by a probe, either radioactive or fluroescent. Once hybridization is complete remove and wash the nitrocellulose sheet, so that only hybridised DNA molecules are left on the sheet. Perform [[autoradiography|autoradiography]] to obtain an image of the hybridised DNA. THis is image can be used to help create a detailed restriction map for this region of the genome <ref>Molecular Biology of the Cell, 5th edition, 2008, B Alberts, p 539</ref>. | |||
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Latest revision as of 14:36, 24 October 2012
Southern blotting, named after its inventor Edwin Southern, is a technique that is used to detect specific DNA sequences using gel-transfer hybridization.
Firstly you have to cut the DNA into smaller strands, this is done using a restriction enzyme. Then you need to perform a gel electrophoresis on agrose gel, using the fragments obtained. The smallest DNA fragments travel the furthest, as they are able to move more easily between the gaps in the agrose gel. Once the gel electrophoresis is complete place a nitrocellulous sheet over the gel, so the DNA fragments are transfered to the sheet. Wash the sheet in an alkaline solution, for example NaOH, this is to ensure that the double stranded DNA is separtaed before hybridization. Place the sheet in a sealed plastic bag thats contains a salt buffered solution and DNA nucleotides and leave in conditions that favour hybridization for a long period of time. Ensure the DNA nucleotides are labeled by a probe, either radioactive or fluroescent. Once hybridization is complete remove and wash the nitrocellulose sheet, so that only hybridised DNA molecules are left on the sheet. Perform autoradiography to obtain an image of the hybridised DNA. THis is image can be used to help create a detailed restriction map for this region of the genome [1].
References
- ↑ Molecular Biology of the Cell, 5th edition, 2008, B Alberts, p 539