title: Escherichia coli 6k oligo microarray
status: Public on Dec 15 2007
contact.city: Vancouver
contact.first: Mickael
contact.last: Le Gac
contact.email: legac@zoology.ubc.ca
contact.address: University Boulevard
contact.country: Canada
contact.institute: University of British Columbia
organism: Escherichia coli
manufacture_protocol: Oligonucleotides were resuspended in 384 well flat-bottom plates (Nunc, New York, USA) to a concentration of 30 μM in 3X SSC (600 pmol of oligo into a volume of 20 μl 3X SSC).  Arrays were printed on EZ-Rays aminosilane slides (Matrix Technologies, Hudson, NH, USA) with the Biorobotics Microgrid II microarray printer (Genomic Solutions, Ann Harbor, Michigan, USA). Microspot 10K quill pins (Biorobotics, Cambridge, UK) in a 48-pin tool were used to deposit 0.5 nL (0.1 ng cDNA) per spot onto the slide. The resulting microarrays have a 12 x12 subgrid layout with 144 features per subgrid, each spot having approximate diameter and pitch of 90 µm and 300µm, respectively. A 280-bp GFP (green fluorescent protein) cDNA was amplified from a GFP clone (BD Biosciences, Mountain View, CA, USA) by using the primers (5’-GAAACATTCTTGGACACAAATTGG-3’) and (5’-GCAGCTGTTACAAACTCAAGAAGG -3’), and printed in subgrid corners to assist in placing on the grid. The slides were crosslinked in a UV Stratalinker 2400 (Stratagene, La Jolla, CA, USA) at 300mJ. All slides are inspected manually to identify any major printing issues. One slide every 20 to 30 slides was hybridized with labeled random 9-mer oligonucleotide (SpotQC, Integrated DNA Technologies, Coraville, IA, USA) or SYBR Green 1 (Invitrogen, Carlsbad, Ca, USA) staining and scanned using ScanArray Express scanner (Perkin Elmer, Wellesley, MA, USA). Presence/absence, shape, signal intensity vs. background, diameter and DNA binding site capability were measured for each spot on the slide using files generated by Imagene software (BioDiscovery Inc., El Secundo, CA, USA). Position and description of flagged spots (spots absent or thought to be unusable during post hybridization analysis) as well as dust, sub-grid defects and other noticed irregularities are recorded. All information collected from all slides controlled for each batch was combined into a report.