title: MHP Human 1Mb_Chr18 CGH array
status: Public on Feb 11 2010
contact.city: Cambridge
contact.first: George
contact.last: Poulogiannis
contact.email: George_Poulogiannis@hms.harvard.edu
contact.address: Tennis Court Road
contact.department: Pathology
contact.country: United Kingdom
contact.institute: University of Cambridge
contact.phone: 001-617-677-8315
organism: Homo sapiens
description: The 1Mb and Chromosome 18 clone set has been assembled by the Mapping Core, Map Finishing and Microarray Facility groups of the Wellcome Trust Sanger Institute, Hinxton, UK. Additional clones were also kindly supplied by the Wellcome Trust Sanger Institute. The array has 312 subarrays (13 rows by 24 columns), each subarray consists of 18 rows by 18 columns.
manufacture_protocol: 1.BAC/PAC clones were grown in 2xTY medium in a deep-well titre plate at 37°C overnight with chloramphenicol 25µg/ml (BAC) or kanamycin 20µg/ml (PAC).
  2. DNA was extracted from BAC or PAC clone using modified alkali-lysis method (microprepping).
  3. Approximately 5ng of BAC/PAC DNA was PCR-amplified using three DOP-primers (Fiegler H, Genes Chromosomes Cancer 36:361-374, 2003).
  4. Two microlitre of the DOP-PCR products was further amplified using 5'-Aminolink universal primer (Fiegler H, Genes Chromosomes Cancer 36:361-374, 2003).
  5. Three volumes of the Amino-PCR products were mixed with one volume of 4xSpotting buffer (1M Sodium phosphate buffer, pH8.5, 0.001% Sarkosyl) to make a spotting mix.
  6. The spotting mix was filtered through Corning 96 well white polystyrene filter plate with modified styrene insert (0.2µm PVDF) by centrifugation.
  7. The filtered spotting mix was transferred to a 384-well plate.
  8. The spotting mix was printed printed in duplicate onto a CodeLink slide (GE Healthcare) using a MicroGrid II robot (Genomic Solutions, Huntingdon, UK) in 4x4 subarrays.
  9. The printed slides were incubated at room temperature for 24-72 hours in a humidified chamber.
  10. The slides were treated with 1% ammonium hydroxide at room temperature for 5 min, then with 0.1% SDS at room temperature for 5min.
  11. The slides were heat-denatured in a boiling water for 2 min, cooled, rinsed with water and dried.