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Method For Quick, Efficient ChIP DNA Purification
Sponsored,vendor-submitted protocol   Published in BioTechniques Protocol Guide 2009 (p.23) DOI: 10.2144/000113040 Abstract

Chromatin immunoprecipitation (ChIP) is an established and useful technique for characterizing cellular protein/DNA associations including those specific for genomic loci having undergone histone modification. Consequently, the purity of ChIP DNA is essential for successful downstream analyses (e.g., PCR, Southern blotting) and accurate data interpretation. The ChIP DNA Clean & Concentrator™ from Zymo Research has been designed for quick (2-minute) recovery of ultra-pure DNA from any chromatin immunoprecipitation (ChIP) assay, cell lysate, Proteinase K–digested sample, PCR mixture, or other enzymatic reaction. Its use in the purification of ChIP DNA derived from yeast cell lysates is demonstrated below.

Chromatin immunoprecipitation (ChIP) is an established and useful technique for characterizing cellular protein/DNA associations including those specific for genomic loci having undergone histone modification. Consequently, the purity of ChIP DNA is essential for successful downstream analyses (e.g., PCR, Southern blotting) and accurate data interpretation. The ChIP DNA Clean & Concentrator™ from Zymo Research has been designed for quick (2-minute) recovery of ultra-pure DNA from any chromatin immunoprecipitation (ChIP) assay, cell lysate, Proteinase K–digested sample, PCR mixture, or other enzymatic reaction. Its use in the purification of ChIP DNA derived from yeast cell lysates is demonstrated below.

Materials

Saccharomyces cerevisiae (strain TMY18)

Anti-RNA Polymerase II IgG (Cat# 05-623; Millipore, Billerica, MA, USA)

Protein A Sepharose™ CL-4B (Cat# 17-0780-01; Amersham, Piscataway, NJ, USA)

ChIP DNA Clean & Concentrator™ (Cat# D5201, D5205; Zymo Research Corporation)

ZymoTaqDNA Polymerase (Cat# E2003; Zymo Research Corporation) Methods ChIP of S. cerevisiae Cell Lysates

  1. Yeast was cultured in YEPD at 30°C. For induction of GAL genes, cells were transferred during log phase growth to YEP medium containing 2% galactose.

  2. Cells were harvested, and then processed according to canonical ChIP procedures including: formaldehyde treatment, cell lysis, and shearing of the DNA.

  3. Cell lysates were immunoprecipitated with an anti-RNA polymerase II antibody and protein A Sepharose™ CL-4B beads.

  4. After reverse cross-linking the samples, the DNA was purified using the ChIP DNA Clean & Concentrator™.

Protocol for the Purification of ChIP DNA Using the ChIP DNA Clean & Concentrator™

  1. In a 1.5 ml tube, add 5 volumes of ChIP DNA Binding Buffer to each volume of sample (5:1). Mix briefly.

  2. Transfer the mixture to a Zymo-Spin™ Column in a Collection Tube.

  3. Centrifuge at ≥10,000× g for 30s. Discard the flowthrough.

  4. Add 200 µl Wash Buffer to the column. Centrifuge for 30 s. Repeat this wash step.

  5. Add 6-100 µl Elution Buffer directly to the column matrix. Transfer the column into a new 1.5 ml tube and centrifuge for 30 s to elute the DNA.

Results

DNA from yeast cell lysates (pre-IP) was isolated using the ChIP DNA Clean & Concentrator™ and analyzed by agarose gel electrophoresis. Figure 1 shows that high quality DNA purified with the ChIP DNA Clean & Concentrator™ was proportional to the sampled cell lysate volume.


Figure 1. Agarose gel electrophoresis of DNA isolated from yeast cell lysates (pre-IP). (Click to enlarge)


Also, PCR and subsequent agarose gel analysis (Figure 2) of purified ChIP DNA showed strong GAL7 and GAL10 signals from cells cultured in YEP with galactose versus cells cultured in YEPD. This demonstrates that following induction of GAL genes, the association of RNA polymerase II with GAL7 and GAL10 promoters is enhanced compared to cells that have not undergone induction. The ChIP DNA Clean & Concentrator™ was demonstrated to be a fast, convenient method for high-quality ChIP DNA recovery/purification.


Figure 2. Yeast ChIP PCR Analysis. (Click to enlarge)


Correspondence

Address correspondence to Zymo Research Corporation, 625 W. Katella Avenue, Suite 30, Orange, CA, 92867, USA; Tel.: 1-714-288-9682; Fax: 1-714-288-9643; www.zymoresearch.com.

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