title: Systemic Analysis of Araboidopsis Promoters (SAP, Benhamed et al. 2008) [Membrane A]
status: Public on Mar 31 2012
contact.city: Gatersleben
contact.first: Astrid
contact.last: Junker
contact.address: Corrensstrasse 3
contact.department: Molecular Genetics
contact.country: Germany
contact.institute: Leibniz Institute for Plant Genetics and Crop Plant Research
organism: Arabidopsis thaliana
manufacture_protocol: For large scale identification of precipitated and amplified chromatin fragments, a macroarray was spotted using 11,904 PCR products comprising upstream regions of Arabidopsis genes which were obtained from the European SAP-project (Systemic analysis of Arabidopsis promoters, (Benhamed et al., 2008). Amplicons were purified by reverse osmosis using MinElute plates (Qiagen, Hilden, Germany) and resuspended in 20µl TE buffer and analyzed on 1% agarose gels before spotting. Approximately 75% of the amplicons appeared as single bands. Amplicons were mixed 1:1 (v/v) with 1M NaOH, 5M NaCl and spotted in duplicate onto positively charged Biodyne B nylon membranes (Pall, Dreieich, Germany) at a density of 140 spots/cm2 using a Microgrid III robot (BioRobotics, Cambrigde, England). After spotting, membranes were washed with 0,5M NaOH, 1,5M NaCl for 5min, neutralized in 1,5M NaCl, 0,5M Tris-HCl, pH 7,4 for 5min, cross-linked in a UV-Stratlinker 2400 (Stratagene, Cedar Creek, United States) at 120mJ and baked for 30min at 80°C in a thermal oven. Amplicons were distributed on two membranes (membrane A and B).