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Dot blot
From Wikipedia, the free encyclopedia
A Dot blot (or Slot blot) is a technique in molecular biology used to detect biomolecules. It represents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by chromatography. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot. This is then followed by detection by either nucleotide probes (for a northern blot and Southern blot) or antibodies (for a western blot).
The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule. Furthermore, if two molecules of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA 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 95°C) and added to the wells where a vacuum sucks the water (with NaOH and NH4OAc) from underneath the membrane (nylon or nitrocellulose).