title: SMCmou8400A Mus musculus 8k cDNA array
status: Public on Jun 09 2007
contact.city: Beaverton
contact.first: Valerie
contact.last: Matagne
contact.email: matagnev@ohsu.edu, valerie.matagne@univ-amu.fr
contact.state: OR
contact.address: 505 nw 185th Ave
contact.department: Neuroscience
contact.country: USA
contact.institute: OHSU
organism: Mus musculus
description: Array format is 16 grids, with four rows (metarows) and four columns (metacolumns) of grids.  Grids are 23 x 23.  Each array was printed twice per slide.  The array contains replicates of the Stratagene SpotReport 10 cDNA validation system.  These are Arabidopsis thaliana genes that have no known homology to mammalian genes.  The labels for these in the genelist are A.thaliana_Cab, A.thaliana_RCA, A.thaliana_rbcL, A.thaliana_LTP4, A.thaliana_LTP6, A.thaliana_XCP2, A.thaliana_RCP1, A.thaliana_NAC1, A.thaliana_TIM, and A.thaliana_PRKase.  Included in the SpotReport material is also human_beta_actin.  Genes not identified in the primarily library listing are noted as NoGeneID.  Locations printed with only buffer are listed as LeftBlank.
manufacture_protocol: 1.  PCR products were derived from the first 88 plates of the NIA 15k Mouse cDNA clone set.  The reference for the library is:  Tanaka TS, et al. (2000). Genome-wide expression profiling of mid-gestation placenta and embryo using a 15000 mouse developmental cDNA microarray. Proc. Natl. Acad. Sci. USA, 97: 9127-9132.
  2.  cDNAs were amplified by PCR using DynaZyme Ext polymerase (MJResearch) using 0.5 units of enzyme per reaction.  Amplification protocol was 94 C for 45 sec, 55 C for 45 sec, and 72 C for 2 min 30 sec for 32 cycles.  Amplifications were performed in an MJResearch PTC-225 Thermal Cycler fitted with four 96-well heads.
  3.  PCR products were separated from unincorporated nucleotides using Millipore Multiscreen PCR filter plates and recovered from the filter in 100 ul of water.  2 microliter aliquots were removed and run on 0.8% agarose gels for quality control. Purified PCR products were reduced from 98 microliters to dryness in a LabConCo CentriVap concentrator.  Dried DNA was resuspended in 30 microliters of 50% DMSO and stored frozen at -20C.
  4.  Three microliters of each PCR product were transferred to Whatman 384-well microtiter plates for printing.  Printing plates were used up to 7 times.  Plates were stored at -20 C between print runs.  Prior to each print run, thawed plates were spun at 3000 rpm in a Sorvall RT7plus tabletop centrifuge for 10 minutes.
  5.  Arrays were printed on Corning UltraGAPS slides using a Cartesian PixSys 5500XL array printer with a Pegasus plate handler attachment.  The plate handler chamber was maintained at 70% relative humidity; the print chamber was maintained at 50% relative humidity.  Printing was done with 16 Telechem CMP-2 array printing pins.  Printing used twenty-two 384-well microtiter plates and took approximately 24 hours.  Printed arrays were baked at 80 C for 3.5 hours in a baking oven.  Arrays were stored in the dark in a sealed desiccator until use.  Prior to use, arrays were blocked for 1 hour at 55 C in 5X SSC, 1.0% SDS, and 2.0% BSA.