title: Escherichia coli 31K spotted oligonucleotide array
status: Public on Jul 25 2009
contact.city: Beijing
contact.first: liu
contact.last: hanhua
contact.address: 18 Life Science Parkway
contact.country: China
contact.institute: CBC
organism: Escherichia coli
manufacture_protocol: The 9, 308 E.coli genome oligo-arrays were constructed by CapitalBio Corp. (Beijing, China) using the 5’-amino-modified 70-mer probe library (Operon, Huntsville, AL). In addition to internal controls, eight intergenic sequences from yeast were used as external controls. All nucleotides were dissolved in DNA Spotting Solution (CapitalBio) at 20 mM and printed on aminosilane-coated glass slides (CapitalBio) using a SmartArrayer-136TM spotter (CapitalBio). After printing, the slides were baked for 1 h at 80°C and stored dry at room temperature until use. Prior to hybridization, the slides were rehydrated over 65°C water for 10 s and UV cross-linked at 250 mJ cm-2 energy. The unimmobilized oligonucleotides were washed off with 0.5% SDS solution for 10 min at 42°C, and SDS was removed by dipping the slides in anhydrous ethanol for 30 s. For hybridization, labeled control and experimental samples were quantitatively adjusted based on the efficiency of Cy-dye incorporation and mixed into 80μl hybridization solution (3×SSC, 0.2% SDS, 25% formamide and 5×Denhart’s). Microarrays were hybridized at 42°C overnight and washed with two consecutive washing solutions (0.2% SDS, 2×SSC at 42°C and 0.2% SSC) for 5 min at room temperature.