title: Cepko BMAP cDNA
status: Public on Jun 01 2006
contact.city: Boston
contact.first: Winston
contact.middle: Patrick 
contact.last: Kuo
contact.email: wkuo@genetics.med.harvard.edu
contact.state: MA
contact.address: 188 Longwood Avenue
contact.department: Genetics
contact.country: USA
contact.institute: Harvard Medical School
organism: Mus musculus
description: E. coli containing the retinal library cDNA inserts were inoculated into 96 deep well blocks containing 1.2 ml of 2xYT media using disposable plastic replicators (Genetix, Hampshire, UK). Cultures were grown at 37°C for 20 hours under constant shaking at 200 RPM and finally pelleted via centrifugation. Plasmids were purified using the QIAprep Turbo kit (Qiagen, Valencia, CA, USA). 96 pin disposable replicators were used to transfer plasmid templates to PCR plates containing 60 ?l of reaction mix. PCR reactions utilizing M13 forward and reverse primers and Taq polymerase (Qiagen) underwent 32 cycles of 95°C for 45 seconds, 53°C for 45 seconds, and 72°C for 2:45. PCR products were purified using QIAquick Kit (Qiagen) and eluted in 100 ?l of water. PCR products were quality scored and sized by running on a 2% agarose 96 well E-gel (Invitrogen Corporation, Carlsbad, CA). Products ranged in size from 300 bp to 3000 bp. The total PCR yield for each well was measured on a SPECTRAmax PLUS spectrophotometer (Molecular Devices, Sunnyvale, CA). Plates containing less than 1.5 ?g of product were reamplified and combined until the total mass exceeded this value. Plates were dried at room temperature, resuspended in 50 ?l of water, and transferred to 384 well V-bottom spotting plates (Genetix).
  
  Spotting plates were dried at room temperature and products were resuspended in 7.5 ?l of 30% DMSO (Sigma-Aldrich, St. Louis, MO, USA) A Biorobotics MicroGrid TAS II with 16 Microspot 10K quill pins (Genomic Solutions, Ann Arbor, MI, USA) was used to array PCR products at a pitch of 260 ?m onto UltraGAPS amino-silane coated slides (Corning, Corning, NY, USA) in a relative humidity of 45% ± 2%. Microarrays were rehydrated in a humidity chamber for 30 seconds, denatured on a heat block at 95°C for 30 sec, and ultimately crosslinked at 125 mJ in a GS Genelinker UV chamber (Biorad, Hercules, CA, USA). Slides were blocked for 1 hour with a 50% formamide, 5x SCC, 0.1% SDS, 0.1 mg/ml BSA solution.
manufacture_protocol: Mouse retinal cDNA clones were assembled from four different libraries generated by M.B. Soares (Bonaldo, Lennon et al. 1996). NIH_BMAP_Ret1 is a normalized library derived from embryonic retina tissue and comprises 9.8% of the collection. NIH_BMAP_Ret2 is a normalized library derived from neonatal retina tissue and comprises 8.8% of the collection. NIH_BMAP_Ret3 is a normalized library derived from adult retina tissue and comprises 8.6% of the collection. NIH_BMAP_Ret4 is a subtracted library derived from retinal tissue at various stages of development and comprises 72.8% of the collection.