It does not involve immobilization of the biomolecules from a gel matrix to the filter membrane.One can detect the presence or absence of genes from the sample of transgenic individuals in a single test run.Dot blot technique does not require the separation of bands on the solid support medium (agarose), or there is no requirement of electrophoresis.Colourimetric detection: Measure the intensity of colour by using the colourimeter.The addition of substrate will give a specific colour to the sample. Add substrate after the attachment of the secondary antibody with the primary antibody. The secondary antibodies attach with the enzymes. Addition of secondary antibody: The secondary antibody specifically binds with the primary antibodies.Washing: After binding the primary antibody with the target protein, wash the filter paper to wash off the unbound primary antibodies by using the PBS buffer.Addition of primary antibody: The primary antibodies fix with the target protein molecule.Blocking: Then, add bovine serum albumin (BSA) medium or dry milk to block the extracellular space in the filter membrane.Blotting: It involves the addition of different protein sample directly onto the nitrocellulose or PVDF filter membrane.Extraction of Protein: Take out different protein samples from different tissues or cells.The identification of Protein by dot blot technique involves the following steps: Autoradiography: Expose the filter membrane to the X-ray film to visualize the target RNA.Washing: After hybridization, wash the unbound or unhybridized radioactive probe from the filter medium.The radioactive probe will complementarily pair with the target RNA or hybridize the RNA. Hybridization: Add the radioactive probe to the filter medium containing the RNA sample.Blotting: It is a second step that involves the blotting of the different RNA sample directly onto the nitrocellulose or nylon filter membrane.Extraction of RNA: Take different samples of the RNA from the different tissues or cells.The identification of RNA by dot blot technique involves the following steps: Autoradiography: Subject the filter membrane to the X-ray film, after which one can visualize the desired gene of DNA.
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Washing: After hybridization, wash the unbound or free radioactive probe from the filter medium.A radioactive probe will bind to the target DNA or later hybridize it. Hybridization: After denaturation, add a radioactive probe to the filter medium containing a DNA sample.Denaturation: The ds-DNA is denatured into the single strands through alkali treatment.Blotting: It is a second step that involves the blotting of the different DNA sample directly onto the nitrocellulose or nylon filter membrane.Extraction of DNA: Take different samples of the DNA from the different tissues or cells.The identification of DNA by dot blot technique involves the following steps: Based on the isolation method of biomolecules like DNA, RNA and protein, A dot blot technique is classified into three types:
The dot blot technique is different for the isolation of DNA, RNA and protein. Dot blot method has an advantage over the other blotting methods, as it does not involve running of the sample on the gel matrix. It can define as the process of identifying biomolecules like DNA, RNA or protein in different samples taken from different cells or tissues of the individuals. Dot blot technique is also called slot blot technique.
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