Cheryl Christianson, Dunham Lab, Princeton University
Extracted molecule
genomic DNA
Extraction protocol
Description : To prepare genomic DNA, cells were grown to saturation in selective media. 15 mls of culture were spun down, rinsed and incubated for 60 minutes at 37oC in 1.5 mls of 1 M Sorbitol, 10 mM Na-phosphate, pH 7.0, 10 mM EDTA, 200 mg/ml zymolase and 150 mM b-mercaptoethanol. Cells were pelleted and incubated in 1.5 mls of 50 mM EDTA, pH 8.0, 0.3% SDS, 200 mg/ml proteinase K and incubated for another 60 minutes at 65oC. 0.6 mls of 5 M KOAc was added and mixture was incubated on ice for 30 min. The supernatant was subjected to a phenol/chloroform extraction and DNA was precipitated. The DNA was RNAse treated at 37oC for 2 hours (10 mM Tri-HCl, 1mM EDTA, pH 7.5, 1 mg/ml RNAse), followed by another phenol/chloroform extraction, and precipitated with ethanol.
Label
Cy3
Label protocol
Description : 1 microg HhaI digested DNA was labeled with Klenow polymerase and Cy3- or Cy5-dCTP according to the BioPrime CGH labeling kit (Invitrogen), using half volume reactions. Yield and dye incorporation were checked with a Nanodrop spectrophotometer.
Cheryl Christianson, Dunham Lab, Princeton University
Extracted molecule
genomic DNA
Extraction protocol
Description : To prepare genomic DNA, cells were grown to saturation in selective media. 15 mls of culture were spun down, rinsed and incubated for 60 minutes at 37oC in 1.5 mls of 1 M Sorbitol, 10 mM Na-phosphate, pH 7.0, 10 mM EDTA, 200 mg/ml zymolase and 150 mM b-mercaptoethanol. Cells were pelleted and incubated in 1.5 mls of 50 mM EDTA, pH 8.0, 0.3% SDS, 200 mg/ml proteinase K and incubated for another 60 minutes at 65oC. 0.6 mls of 5 M KOAc was added and mixture was incubated on ice for 30 min. The supernatant was subjected to a phenol/chloroform extraction and DNA was precipitated. The DNA was RNAse treated at 37oC for 2 hours (10 mM Tri-HCl, 1mM EDTA, pH 7.5, 1 mg/ml RNAse), followed by another phenol/chloroform extraction, and precipitated with ethanol.
Label
Cy5
Label protocol
Description : 1 microg HhaI digested DNA was labeled with Klenow polymerase and Cy3- or Cy5-dCTP according to the BioPrime CGH labeling kit (Invitrogen), using half volume reactions. Yield and dye incorporation were checked with a Nanodrop spectrophotometer.
Hybridization protocol
Description : 200 ng differentially labeled DNA from the reference strain and the strain of interest were mixed, combined with control targets and hybridization buffer, boiled for 5 minutes, and applied to a microarray consisting of 60mer probes for each yeast open reading frame (Agilent). Microarrays were rotated at 60?C for 17 hours in a hybridization oven (Agilent). Arrays were then washed according to the Agilent SSPE wash protocol.
Scan protocol
Pixel Size : 10 Scan Date : 2006-07-21 Scan Time : 10:50:14 Scanner Make : Agilent Technologies Scanner Scanner Model : G2505B Scanning software : ChipScan Scanning software version : A.6.3.1
Description
former name: CC 11311 vs. 12689 H+W DNA
Data processing
Extraction Software : Agilent Feature Extractor Extraction Software Version : A.7.5.1 Datafile type : Agilent result file
