Wet-lab portion of the workflow

Wet-lab portion of the workflow
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  • In step one, the protein of interest (POI) is go-linked with the DNA website it binds to in an in vitro surroundings. Usually this is completed via a mild formaldehyde fixation this is reversible with warmth.
  • Then, the cells are lysed and the DNA is sheared by means of sonication or using micrococcal nuclease. This effects in double-stranded chunks of DNA fragments, commonly 1 kb or less in duration. Those that have been cross-linked to the POI shape a POI-DNA complicated.
  • In the following step, only these complexes are filtered out of the set of DNA fragments, the usage of an antibody particular to the POI. The antibodies may be connected to a strong floor, can also have a magnetic bead, or a few other physical assets that lets in separation of move-connected complexes and unbound fragments. This method is largely an immunoprecipitation (IP) of the protein. This can be carried out either by using using a tagged protein with an antibody against the tag (ex. FLAG, HA, c-myc) or with an antibody to the local protein.
  • The pass-linking of POI-DNA complexes is reversed (generally through heating) and the DNA strands are purified. For the rest of the workflow, the POI is now not essential.
  • After an amplification and denaturation step, the unmarried-stranded DNA fragments are categorized with a fluorescent tag inclusive of Cy5 or Alexa 647.
  • Finally, the fragments are poured over the surface of the DNA microarray, which is noticed with short, single-stranded sequences that cowl the genomic part of interest. Whenever a categorized fragment “unearths” a complementary fragment at the array, they will hybridize and form once more a double-stranded DNA fragment.

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