title: Human 1 Mb - resolution CGH array
status: Public on Apr 18 2014
contact.city: Stockholm
contact.first: Anna
contact.last: Kwiecinska
contact.email: anna.kwiecinska@ki.se
contact.address: CCK R8:00
contact.department: Oncology-Pathology
contact.country: Sweden
contact.institute: Karolinska Institute
organism: Homo sapiens
description: The 1Mb clone set has been assembled by the Mapping Core, Map Finishing and Microarray Facility groups of the Wellcome Trust Sanger Institute, Hinxton, UK. The array has 24 subarrays (6 rows by 4 columns), each subarray consisted of 18 rows by 18 columns.
manufacture_protocol: 1. BAC/PAC clones were grown in 2 x TY medium in a deep-well titrate plate at 37° overnight with chloramphenicol 25 µg/ml (BAC) or kanamycin 205 µg/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 futher 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 4 x Spotting buffer (1 M 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 (0.2 µm PVDF) by centrifugation.
  7. The filtered spotting mix was transferred to a 384-well plate.
  8. The spotting mix was printed in duplicate onto a CodeLink slide (GE Healthcare) using MicroGrid II robot (Genomic Solutions, Huntingdon, UK) in 4 x 4 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 5 min.
  11. The slides were heat-denaturated in a boiling water for 2 min, cooled, rinsed with water and dried.