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qPCR of Yeast ChIP DNA
Published: Sep 20, 2011 DOI: 10.21769/BioProtoc.135 Views: 17212
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Abstract
This protocol is adapted from Chris Yellman's personal notes for qPCR of yeast ChIP DNA to verify binding sites already identified by microarray/sequencing analysis. In addition, qPCR is a quick way to assay the quality of a ChIP sample (provided you have a few well-characterized binding sites) before submitting it for sequencing/microarray analysis. This protocol is designed for use with a Roche LightCycler.
Materials and Reagents
- PCR primers
a. Prepare 10 µM primer stocks in water from 100 µM stocks in TE.
b. Pick several positive control primer pairs and at least one negative site. Primer design criteria are described in the protocol. - LightCycler 480 SYBR Green I Master mix (Roche Diagnostics, catalog number: 04707 516001 )
The mix contains Taq, dNTPs and the intercalating SYBR Green dye. - Yeast genomic DNA for a positive control dilution series
You can use a glass bead lysis yeast genomic DNA preparation that has been RNase treated, Qiagen cleaned and stored in TE, or purchase directly from Invitrogen. - 10 µl capacity multi-well pipetter
384-well reaction plate with transparent adhesive plastic cover
Equipment
- Covered container
- Microcentrifuge tube
- 384-well reaction plate
Procedure
Category
Microbiology > Microbial genetics > RNA > qRT-PCR
Biochemistry > Protein > Immunodetection > ChIP
Molecular Biology > RNA > qRT-PCR
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