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Dot blot (or Slot blot) is a technique in molecular biology used to detect biomolecules. It replaces either northern blot, Southern blot or western blot. In dot blot the biomolecules to be detected are not separated by chromatography. Instead a mixture possibly containing the molecule to be detected is applied directly on a membrane as a dot. This is then immediately followed by detection by either nucleotide probes (northern blot and Southern blot) or antibody (western blot).
Additional recommended knowledge
The technique offers significant savings in time as chromatography and complex blotting procedure for the chromatography gel are not required. However, it offers no information on the size of the biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single blot. Dot blot therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the probes or the antibody.
A radioactive sample can be hybridized to it allowing the researcher to detect variation between samples. The DNA is quantified and equal amounts are aliquoted into tubes in excess of the number of its targets in the samples, such as 10ug for a plasmid and 1ug for a PCR amplicon. These are denatured (NaOH and 95C) and added to the wells where a vacuum sucks the water (with NaOH and NH4OAc) from underneath the membrane (nylon or nitrocellulose).
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Dot_blot". A list of authors is available in Wikipedia.|