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Yeast Transcription Factor Chromatin Immunoprecipitation
Published: Jul 5, 2011 DOI: 10.21769/BioProtoc.92 Views: 13359
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Abstract
This ChIP protocol was developed and improved over the years by various researchers in the Snyder lab, Stanford University, especially Anthony Borneman and Christopher Yellman. I have used this method to successfully map the genome-wide binding of transcription factors Ste12. The ChIPed DNA is suitable for downstream analysis using PCR, microarray or sequencing.
Materials and Reagents
- 500 ml of log-phase yeast cell culture per ChIP (at -0.9 x 107 cells/ml, ~4.5 x 109 cells per sample)
- 37% formaldehyde
- 2.5 M glycine in H2O (heat sterilized)
- Liquid nitrogen, dry ice/ethanol bath or -70 °C freezer
- 0.5 mm Zirconia/Silica Beads (Bio Spec Products, catalog number: 11079105z )
- Commercial protease inhibitor cocktails, for example:
Roche Complete protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11697498001 )
Roche Complete Mini protease inhibitor cocktail tablets (F. Hoffmann-La Roche, catalog number: 11836153001 ) - EZview anti-Myc affinity gel (red colored beads) (Sigma-Aldrich, catalog number: E6654 )
- Minelute kit for final DNA purification (QIAGEN, catalog number: 28004 )
- LiCl
- NaOAc
- Ethanol
- Triton X-100
- TE
- NaCl
- EDTA
- Isopropanol
- NP-40
- Na-deoxycholate
- SDS
- Tris-buffered saline (TBS) (10x stock) (see Recipes)
- Lysis/IP buffer (see Recipes)
- Lysis buffer/500 mM NaCl (see Recipes)
- IP wash solution (see Recipes)
- TE/1% SDS (100 ml) (see Recipes)
- TE/0.67% SDS (100 ml) (see Recipes)
- TE (100 ml) (PH 8.0) (see Recipes)
- 1 mM PMSF (Fluka, catalog number: 93482 ) (see Recipes)
Equipment
- Millipore stericup sterile vacuum filter units, 500 ml funnel, 0.22 μm or 0.45 μm pore size (EMD Millipore, catalog number: SCGVU05RE , SCHVU11RE )
- Syringe needle (BD Biosciences, catalog number: 305155 or 305156 )
- 5 ml snap-cap tubes, polypropylene (preferred) or polystyrene (BD Biosciences, Falcon®)
- 15 and 50 ml conical polypropylene screw-top tubes (BD Biosciences, Falcon®)
- Branson Sonifier 250 with microtip or Digital Sonifier S-450D (BD Biosciences)
- Refrigerated tabletop centrifuges, e.g. Beckman GS-6R, GS-15R (Beckman Coulter), or Eppendorf refrigerated multipurpose centrifuges (EppendorfTM, model: 5810R and 5804R ) (or simply put an ordinary tabletop centrifuge in the cold room)
- Fume hood
- FastPrep machine (FastPrep, catalog number: 6004500 )
- Hemacytometer
- Spectrophotometer
Procedure
Category
Molecular Biology > DNA > DNA-protein interaction
Microbiology > Microbial genetics > DNA
Biochemistry > Protein > Immunodetection
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