title: ACRF microarray consortium human 8k array
status: Public on Dec 01 2005
contact.city: Parkville
contact.first: Ken
contact.last: Simpson
contact.email: ksimpson@wehi.edu.au
contact.state: Victoria
contact.address: 1G Royal Parade
contact.department: Genetics and Bioinformatics
contact.country: Australia
contact.institute: The Walter and Eliza Hall Institute
contact.phone: +61-3-93452628
organism: Homo sapiens
description: H8K210302, barcodes 1719-1774
  Spotted
  Corning GAPS-II Amino silane Slides
  25x75mm Slides
  18432 dots
  Custom made with human 1K at end
  Reporter Type: PCR products of cDNA clones, Double stranded
  Primer  sequence used to amplify human 8K clone set plates 1-75, and Anthea's 1K human plates:
  Reverse 5'gtgagcggataacaatttcacacaggaaacagc3'
  Forward 5'ctgcaaggcgattaagttgggtaac 3'
  Primer sequence used to amplify human 8K clone set plates 76-78:
  Reverse 5'cacaggaaacagctatgacca3'
  Forward 5'acggccagtgccagcttat3'
  cDNA Clone set from Research Genetics, Alabama, USA
  Human 8K clone set obtained Nov 2001, Housed at AGRF, Melbourne Australia
  Anthea's 2 plates leukemia subset of human 40K clone set obtained Sep 2000, housed at AGRF, Melbourne Australia.
  Amplification Protocol: (Human 8K only)
  0.2mM dNTP, 25 pmols each primer, 2.5 units HotStar Qiagen TAQ, 1X HotStar buffer, and 1ul straight clone culture in 100ul reactions.
  95C for 15min; 7 cycles of 94C for 30s; 72C for 1min (-1C every cycle); 72C for 2min; 23 cycles of 94C for 30s; 65C for 1min; 72C for 2min; final elongation 72C for 5min; 4C 2min.
  Amplification Protocol: (Anthea's plates)
  95C for 15min; 38 cycles of 94C for 30s; 60C for 45s; 72C for 3.5min; final elongation 72C for 5min; 4C 2min.
  Millipore PCR plate purification, gel analysis, aliquot into 384 well plates by robot (A1,A2,B1,B2).
  Resuspended in water for 4 days in fridge, then added an equal amount of DMSO.
  Printing concentration was ~250 ng/ul in 50% DMSO, 6ul total.
  Post Processing: UV crosslinked 70mj; baked 2hrs at 80C; stored dessicated.
  Feature Information:
  ~150um diameter
  Covalent and ionic attachment.
  Control Elements on the array:
  Human 8K control plate version 1, 6ul 50%DMSO
  Printing Buffer and blank wells for pin washing controls
  Various dilutions of geographic control (concentrated mix of all PCR products in that library set) for concentration, normalisation and positive controls.
  Various mouse, human and yeast 'housekeeping genes'.
  Poly A, pGEM, Human Cot1 DNA, Mouse Cot1 DNA, salmon sperm are all spotted to assess sufficient blocking of repetitive elements which if not properly blocked can give rise to non-specific annealing.
  Printed controls for future spiked (exogenous) applications (yeast intergenic) SP2, SP3; present negative controls
  RT efficacy controls based on yeast intergenic RT1-7, again for future, presently negative controls.