Wash cells twice in 25 ml ice-cold PBS (pH 7.4). Discs/pellets are then washed in 10 ml of 0.5 M glycine for 5 minutes before the biomass is harvested. Cross-link proteins to DNA: Wash pellet or submerge disc in 10 ml of a 1% (v/v) formaldehyde solution for 20 minutes at room temperature to cross-link proteins to DNA.Optional: At this point, a small sample can also be taken for western blot to confirm expression of the desired protein using an antibody that is specific to the protein of interest of the tag. For sampling, harvest liquid cultures by centrifugation or removing cellophane discs and biomass from the plate at the desired time point. Sampling: Cultures can be grown in either liquid culture or on solid agar plates (on cellophane discs).Elution Buffer – 50mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS.TE Buffer – 10 mM Tris-HCl pH 8.0, 1 mM EDTA.IP Buffer – 100 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% v/v Triton-X.Lysis Buffer – 10 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mg/ml lysozyme.a 3x FLAG-TAG) to allow for immunoprecipitation of the protein-DNA complexes. Alternatively, a strain expressing the protein of interest with a tag fused to one terminus (e.g.
In order to carry out a ChIP-Seq experiment, a specific antibody for the protein of interest is required. Any DNA that is bound to the protein of interest can then be identified by sequencing in order to characterise the DNA binding sites for the protein of interest. It works by cross-linking proteins to DNA, shearing the DNA and then pulling down the protein of interest. Chromatin Immunoprecipitation and sequencing can be used to characterise protein-DNA interactions.
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