Summary: Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1975. It allows investigators to determine the molecular weight of a restriction fragment, to measure relative amounts in different samples and to locate a particular sequence of DNA within a complex mixture. DNA (genomic or other source) is digested with a restriction enzyme and separated by gel electrophoresis and transferred from an agarose gel onto a membrane which is then incubated with a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe is labeled before hybridization either radioactively or enzymatically (e.g. alkaline phosphatase or horseradish peroxidase). Finally, the location of the probe is detected by directly exposing the membrane to X-ray film or chemiluminescent methods.
Subcategories
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Protocols
- Southern Blot (Dr. Volker Briese, Universitäts-Frauenklinik Rostock)
Localization of particular sequences within genomic DNA is usually accomplished by the transfer techniques described by Southern (1975). Genomic DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through an agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually nitrocellulose filter or nylon membrane). The relative positions of the DNA fragments are preserved during their transfer to the filter. The DNA attached to the filter is hybridized to radiolabeled DNA or RNA, and autoradiography is used to locate the positions of bands complementary to the probe.
http://www-ufk.med.uni-rostock.de/lablinks/protoco...
Added: Sat Nov 02 2002, Hits: 1663, Reviews: 0 Write review Cached - Southern Blotting (Gottschling Lab,Fred Hutchinson Cancer Research Center )
Sourthern protocol describes DNA transfer, hybridization, washing and stripping...
http://www.fhcrc.org/labs/gottschling/misc/souther...
Added: Fri Jul 26 2002, Hits: 2600, Reviews: 2 Read reviews Write review Cached -
Alkaline Southern Blotting
(Kim Marshall, Dendrome)
http://dendrome.ucdavis.edu/Protocols/alkalineblot...
Added: Tue May 14 2002, Hits: 586, Reviews: 0 Write review Cached -
Analysis of Genomic DNA by Southern Hybridization
(Jun-Ryul Huh)
Great protocol for Southern including Probe preparation, filter preparation, filter suppression (pre-hybridization), probe suppression, washing and exposure
http://www.tree.caltech.edu/protocols/genomic_sout...
Added: Tue May 14 2002, Hits: 1057, Reviews: 0 Write review Cached -
DNA Hybridization and Detection by Chemiluminescence
(Janos Luka Ph.D., Department of Pathology, Eastern Virginia Medical School)
Describes Southern transfer, hybridization and chemiluminescence detection with recipes.
http://herpesvirus.tripod.com/research/DNAhyb.htm
Added: Fri Apr 16 2004, Hits: 400, Reviews: 0 Write review Cached -
Hybridization and Detection of Dig-Labeled Probes
(CIMMYT Applied Molecular Genetics Laboratory)
These protocols have been optimized for hybridizations in siliconized glass bottles (e.g. Robbins Scientific Corp. or similar) and in polypropylene Corning tubes.
http://www.cimmyt.org/ABC/Protocols/HybridDetectio...
Added: Mon Aug 05 2002, Hits: 606, Reviews: 0 Write review -
Hybridization to Southern Blots
(Donis Keller Lab)
To hybridize radioactively labeled DNA probes to Southern blots containing human DNA to detect Restriction Fragment Length Polymorphosims (RFLPs). This method can also be used for most other applications (e.g., slot blots, zoo blots, yeast YAC blots etc.).
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 672, Reviews: 0 Write review Cached -
Labeling DNA with 32P by Primer Extension
(Donis Keller Lab)
Double stranded DNA is heat-denatured and random hexanucleotide primers are attached to the single strand. The synthesis is carried out using Klenow fragment of DNA polymerase I incorporating 32P-dCTP at the same time.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 333, Reviews: 0 Write review Cached -
Making Combination Markers for Size S
( )
tandards on Southern Blots(Donis Keller Lab)
Digesting lambda DNA (BRL) with a specific restriction enzyme will result in lambda DNA fragments of known size. Three separate lambda digests (BglII, BstEII, and XhoI) will give 23 fragments with a molecular weight range of 60 bp to 33,498 bp (see illustration below). The three digests are pooled, diluted, and used as molecular weight size standards for Southern transfers.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 113, Reviews: 0 Write review Cached -
Making Combination Markers for Size Standards on Southern Blots
(Donis Keller Lab)
Digesting lambda DNA (BRL) with a specific restriction enzyme will result in lambda DNA fragments of known size. Three separate lambda digests (BglII, BstEII, and XhoI) will give 23 fragments with a molecular weight range of 60 bp to 33,498 bp (see illustration below). The three digests are pooled, diluted, and used as molecular weight size standards for Southern transfers.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 144, Reviews: 0 Write review Cached -
Prehybridization of Repeat-Containing Clones with Total Human DNA
(Donis Keller Lab)
To reduce the lane background due to the repetitive sequences, large amount of unlabeled human DNA is added to the labeled probes immediately before the denaturing and hybridizing steps...
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 103, Reviews: 0 Write review Cached -
Preparation of Sonicated Human DNA
(Donis Keller Lab)
To break up high molecular weight human placental DNA into fragment sizes of 500 bp or less which can be used as competitor DNA in Southern and in situ hybridizations. This method can also be used for sonicating salmon sperm DNA.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 114, Reviews: 0 Write review Cached -
Primer Extension Labeling of DNA in Low Melt Agarose
(Donis Keller Lab)
This procedure allows a restriction fragment(s) to be labeled directly from a gel slice, without the associated losses of DNA usually seen with gel purification methods.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 112, Reviews: 0 Write review Cached -
Southern Analysis of Mouse Toe/Tail DNA
(Bowtell Lab Manual, Peter MacCallum Cancer Institute)
http://www.pmci.unimelb.edu.au/core_facilities/man...
Added: Tue May 14 2002, Hits: 304, Reviews: 0 Write review Cached -
Southern Blot
(Bowtell Lab)
http://grimwade.biochem.unimelb.edu.au/bowtell/mol...
Added: Tue May 14 2002, Hits: 847, Reviews: 0 Write review Cached -
Southern Blot
(Bowtell Lab Manual, Peter MacCallum Cancer Institute)
http://www.pmci.unimelb.edu.au/core_facilities/man...
Added: Tue May 14 2002, Hits: 572, Reviews: 0 Write review Cached -
Southern blot-chemiluminescent detection, using CSPD
(NWFSC)
http://micro.nwfsc.noaa.gov/protocols/southern-csp...
Added: Tue May 14 2002, Hits: 239, Reviews: 0 Write review -
Southern blot-chemiluminescent detection, using Lumigen or Lumiphos
(NWFSC)
Non-radioavtive detection
http://micro.nwfsc.noaa.gov/protocols/southern-csp...
Added: Tue May 14 2002, Hits: 176, Reviews: 0 Write review -
Southern Blotting
(Corbett Lab)
http://www.biochem.emory.edu/labs/acorbe2/protocol...
Added: Tue May 14 2002, Hits: 613, Reviews: 0 Write review -
Southern blotting
(Julie B. Wolf, University of Maryland, Baltimore County)
http://www.research.umbc.edu/~jwolf/m11.htm
Added: Tue May 14 2002, Hits: 519, Reviews: 0 Write review Cached -
Southern Blotting onto Non-Charged Membranes
(CIMMYT Applied Molecular Genetics Laboratory)
http://www.cimmyt.org/ABC/Protocols/SouthernBlotNo...
Added: Mon Aug 05 2002, Hits: 239, Reviews: 0 Write review -
Southern Hybridization
(ASIRC)
http://molbio.cbs.umn.edu/asirc/protocol/southern....
Added: Tue May 14 2002, Hits: 319, Reviews: 0 Write review Cached -
Southern Hybridization
(Pat Heslop-Harrison and Trude Schwarzacher, Department of Biology, University of Leicester)
Two protocols are given. The first one is used for BAC library screening on filters, although is also suitable for transfer hybridizations.
http://www.le.ac.uk/biology/phh4/methods/southern....
Added: Sat Mar 27 2004, Hits: 197, Reviews: 0 Write review Cached -
Southern Hybridization
(Laurie Lab, University of Virginia Health Science Center)
Detailed protocol for for analysis of genomic DNA or ordering of clones.
http://www.people.virginia.edu/~gwl6s/protocols/so...
Added: Tue May 14 2002, Hits: 441, Reviews: 0 Write review Cached -
Southern Transfer to Immobilon-N
(Donis Keller Lab)
Immobilon- N is a non-nylon membrane that can be used very much like Zetabind in Southern transfers. Because it is extremely hydrophobic when dry, the membrane must first be prewetted in alcohol prior to use.
http://wheat.pw.usda.gov/~lazo/methods/donis/south...
Added: Tue May 14 2002, Hits: 160, Reviews: 0 Write review Cached