1 Method 2 Result 3 Applications 4 See also 5 References 6 External links
Restriction endonucleases are used to cut high-molecular-weight DNA
strands into smaller fragments.
The DNA fragments are then electrophoresed on an agarose gel to
separate them by size.
If some of the DNA fragments are larger than 15 kb, then prior to
blotting, the gel may be treated with an acid, such as dilute HCl.
This depurinates the DNA fragments, breaking the DNA into smaller
pieces, thereby allowing more efficient transfer from the gel to
If alkaline transfer methods are used, the DNA gel is placed into an
alkaline solution (typically containing sodium hydroxide) to denature
the double-stranded DNA. The denaturation in an alkaline environment
may improve binding of the negatively charged thymine residues of DNA
to a positively charged amino groups of membrane, separating it into
single DNA strands for later hybridization to the probe (see below),
and destroys any residual RNA that may still be present in the DNA.
The choice of alkaline over neutral transfer methods, however, is
often empirical and may result in equivalent results.
A sheet of nitrocellulose (or, alternatively, nylon) membrane is
placed on top of (or below, depending on the direction of the
transfer) the gel. Pressure is applied evenly to the gel (either using
suction, or by placing a stack of paper towels and a weight on top of
the membrane and gel), to ensure good and even contact between gel and
membrane. If transferring by suction, 20X
SSC buffer is used to ensure
a seal and prevent drying of the gel. Buffer transfer by capillary
action from a region of high water potential to a region of low water
potential (usually filter paper and paper tissues) is then used to
move the DNA from the gel onto the membrane; ion exchange interactions
bind the DNA to the membrane due to the negative charge of the DNA and
positive charge of the membrane.
The membrane is then baked in a vacuum or regular oven at 80 °C
for 2 hours (standard conditions; nitrocellulose or nylon membrane) or
exposed to ultraviolet radiation (nylon membrane) to permanently
attach the transferred DNA to the membrane.
The membrane is then exposed to a hybridization probe—a single DNA
fragment with a specific sequence whose presence in the target DNA is
to be determined. The probe DNA is labelled so that it can be
detected, usually by incorporating radioactivity or tagging the
molecule with a fluorescent or chromogenic dye. In some cases, the
hybridization probe may be made from RNA, rather than DNA. To ensure
the specificity of the binding of the probe to the sample DNA, most
common hybridization methods use salmon or herring sperm DNA for
blocking of the membrane surface and target DNA, deionized formamide,
and detergents such as SDS to reduce non-specific binding of the
After hybridization, excess probe is washed from the membrane
(typically using SSC buffer), and the pattern of hybridization is
Hybridization of the probe to a specific DNA fragment on the filter
membrane indicates that this fragment contains
^ Southern, Edwin Mellor (5 November 1975). "Detection of specific
sequences among DNA fragments separated by gel electrophoresis".
Journal of Molecular Biology. 98 (3): 503–517.
doi:10.1016/S0022-2836(75)80083-0. ISSN 0022-2836.
^ Towbin; Staehelin, T; Gordon, J; et al. (1979). "Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications". PNAS. 76 (9): 4350–4.
doi:10.1073/pnas.76.9.4350. PMC 411572 .
^ Burnette, W. Neal (April 1981). "Western Blotting: Electrophoretic
Transfer of Proteins from Sodium Dodecyl Sulfate-Polyacrylamide Gels
Library resources about Southern blot
Resources in your library Resources in other libraries
v t e
Southwestern blot (protein:DNA)
Electrophoretic mobility shift assay