title: SMChum8400A cDNA Array
status: Public on Jul 17 2007
contact.city: Beaverton
contact.first: Alejandro
contact.last: Lomniczi
contact.email: lomniczi@ohsu.edu
contact.state: OR
contact.address: 505 NW 185th Ave
contact.department: Neuroscience
contact.country: USA
contact.institute: Oregon National Primate Research Center - OHSU
contact.phone: 503-690-5305
organism: Homo sapiens
manufacture_protocol: 1.  PCR products were derived from the first 88 plates of the Research Genetics distribution of the I.M.A.G.E. Consortium Human cDNA library.  The link for the I.M.A.G.E. Consortium is http://image.llnl.gov/.
  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.