Vacuum blotting also uses a flow of buffer to elute bands onto the membrane, but it uses a vacuum instead of capillary action to create the flow. Generally, 80-90% of the molecules in the gel can be recovered on the membrane by this procedure. Conditions are chosen such that binding to the membrane is favored, so molecules of nucleic acid are trapped there. As buffer flows into the gel from the tank below, the nucleic acids in the gel are carried upward, until they meet the membrane. During this period, the buffer is wicked out of the gel through the membrane and onto the dry filters above. A weight is placed on top of the stack to ensure continued close contact of all components, and the entire assembly is left to stand for 12-16 hours (i.e. The membrane is placed on top of the gel, and a stack of absorbent paper is placed over it. The gel is placed on porous support (generally filter paper or a sponge) which holds the gel above the buffer while allowing the buffer to flow up to it. Electroblotting is also used occasionally, although it requires special care to prevent the crushing or melting of the agarose gel.Ĭapillary flow transfers are carried out in a dish of buffer (see figure below). The two most common methods used for Northern and Southern blotting are capillary flow and vacuum transfer. The flow of buffer elutes the nucleic acid molecules from the gel onto the membrane, preserving the band pattern.īlots are created by laying a membrane over one face of the gel and then creating a flow that carries the molecules in the gel onto the membrane. Transfer buffer is drawn up the wick, through the gel and membrane, and into the dry stack of towels. In all cases the advantage gained by blotting onto a membrane was the immobilization of the electrophoretic pattern, rendering the molecules in that pattern accessible to macromolecular probes. Soon thereafter RNA was blotted successfully (Northern Blotting) and protein ( Western Blotting). The result dubbed a "Southern Blot", reveals a pattern of bands showing the size and relative amount of DNA molecules containing the probe sequence. Southern demonstrated that DNA could be electrophoretically fractionated, transferred to nitrocellulose, and then probed with radioactively labeled DNA sequences, which would hybridize to their cognates bound to the membrane. This process, blotting, was first publicized by Southern (1975). In order to circumvent this problem, a method was devised to "print" an electrophoretic pattern onto a solid support, preserving the positional information from a gel, but removing the matrix. Only complementary fragments would be "stained." However, specific hybridization requires nucleic acid polymers of twenty-five or more bases, which are too large to diffuse rapidly into a gel. In theory, labeled nucleic acid molecules could act as specific "stains" for DNA or RNA species in gels. This is the basis of the RNase protection assay, and PCR amplification, among other techniques. A labeled nucleic acid molecule of known sequence can facilitate detection of any complementary molecules in an unknown sample.
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