Technique / Molecular Biology / DNA analysis techniques / DNA southern blot

Southern blot hybridization is named after its inventor, the British biologist Edwin Southern.

DNA southern blot hybridization new protocol
Southern blot hybridization is one of the most commonly used molecular biology techniques to detect Specific DNA sequences using labeled probes. The DNA southern blot hybridizatin is illustrated bellow: ...

Analys of Genomic DNA by Southern Hybridization (Southern Blot) protocol
From Heiner Müller lab. 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 s ...

PCR Southern blot hybridization using oligonucleotide probe new protocol
PCR Southern blot hybridization using oligonucleotide probe: 1. Capillary transfer DNA (PCR product) on nylone membrane overnight. 2. Neutralize membrane in 0.5M Tris.HCl + 1.5M NaCl for 30 min. 3. Dry membrane. 4. Fixation under UV-lamp. (less than 1min ...

Overview on DNA southern blot hybridization with figures review
This is a brief overview of how a Southern blot (more formally called an DNA blot) is performed and what type of data you can obtain form one. It includes a nice figure which shows how well the DNA digestion should be. ...

Southern blot background troubleshooting
Some common causes for high background in southern blot hybridization: 1. High concentration of probe (should not exceed 5-10ng/ml). Probe sequence too short (for oligonucleotide probes). Probes not denatured properly. 2. Low efficient purification of labe ...

Southern blot membrane cross-link troubleshooting
A friend of mine wants me to find out if nylon membranes can be baked without a vacuum to cross-link DNA onto the membranes. WE do it all the time! Seems to work in a regular oven just fine. This has been said before (sorry, don't rememb ...

Digested DNA migration pattern on agarose gel for southern blot troubleshooting
Southern blot digested DNA abnormal migration pattern on agarose gel: I get pretty good quality DNA from mouse tails (using phenol/cia) to use for Southen blots. I digest the DNA in a large volume and then precipiate it in NaCl/ethanol. When I run th ...

Genomic DNA Southern blot hybridization Alkaline Transfer Problem troubleshooting
I am performing downward alkaline transfer for genomic DNA blot hybridizations (and the transfers are great!), however, the hybridization signal is just not there. I was wondering whether or not the alkaline conditions (pH ~10-11) after the transfer ( ...

Plasmid vector unspecific hybridization troubleshooting
I am screening a plasmid cDNA library of the liver (pSport-vector). After picking the positives I do minipreps, cut out the inserts from the vector by RE-digestion and separate by agarose gel electrophoresis. The blotted gel is hybridized and washed u ...

Protein blocking solution for DNA hybridization troubleshooting
I routinely use the Boehringer Mannheim Blocking Reagent (sold as part of the DIG system) as part of the prehybridisation/hybridisation mix when doing 32-P-labelled Northern and Southern blots, as this avoids the use of (expensive, time consuming) den ...

Hybridization buffer recipe troubleshooting
Does anybody out there know a successful recipe of hybridization buffer without the denatured salmon sperm DNA for genomic southern hybridization? Thanks. XY Zheng ...

Digoxigenin DIG Southern Blots troubleshooting
Does anyone out there have experience with using Boehringer-Mannheim's digoxigenin-labeled (DIG-labeled) oligonucleotide probes to detect target genomic DNA on their Southern blots. I am getting good transfer of the genomic DNA to a nylon membrane. I'm having ...

High background in Southern hybridization hot troubleshooting
I've had annoying problems with high background in standard Southern hybridization. I have digested 5 ug of human genomic DNA w/ EcoRI, BamHI and HindIII and used several different probes (500 bp, no Alus or other repeats, labeling with Amersham RediP ...

Gene copy number by slot-blot hybridization troubleshooting
I'm planning to use slot blot hybridization to calculate the gene copy number in the genome. However, my slot blot give a very weak signal even 50ug of genomic DNA was applied. Is there any possible casue for this..?? I am using DIG labelling an ...

Southern analysis with genomic fragment troubleshooting
I have to use a large fragment of DNA from a gen clone as a probe for a Southern blot. I know it contain some repititive elementss. Is there a protocol for pre-annealing the labelled DNA fragemn to block the repeatst prior to hybridisation? Many thank ...

DNA hybridization using 100 bp insert as probe troubleshooting
I have a question concerning DNA probes for southern or northern blots. I have a 100 bp insert in Bluescript that I wish to label as a probe. Since I want to random prime the insert, I wish to cut out a 500 bp fragment, that is, the fragment will cont ...

southern enigma troubleshooting
I have a 600bp DNA fragment which I cut out of a plasmid, purified from agarose gel with Gene Clean and labeled with 32P using random prime method. I then used the probe to screen a lamba ZAP cDNA library (Stratagene) and isolated several clones. I is ...

Southern Hybridization Strigency Question troubleshooting
A few general questions first: In Southern hybridization experiments how important is hybridization temperature? What are the effects of rasing or lowering the hybridization temperature? How does one determine the proper washing condition ...

Sensitivity of DIG-Southern? troubleshooting
I was trying to set up a non-radioactive Southern to confirm transgenic plants. I used a DIG-labelled NPTII probe (PCR DIG probe synthesis kit from Boehringer Mannheim; 2 µl probe per ml hybridization buffer) and 10 µg of digested genomic arabido ...

Southern blot smear caused by long terminal repeats LTR new troubleshooting
A pdf troubleshooting on southern blot smear caused by MaLR mammalian apparaent long terminal retro-elements. (University of Toronto) ...

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Southern blotting.
(Curr Protoc Mol Biol. 2001 May;Chapter 2:Unit2.9A.)
Brown T.
University of Manchester Institute of Science and Technology, Manchester, United Kingdom.

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit presents several protocols for DNA transfer to nylon and nitrocellulose membranes, and for calibrating a UV transilluminator to cross-link the transferred DNA to the membrane.