title: qChipPre BAC Version
status: Public on Jan 01 2014
contact.city: BARCELONA
contact.first: Lluis
contact.last: Armengol
contact.state: Catalonia
contact.address: Doctor Aiguader, 88
contact.country: Spain
contact.institute: qGenomics
organism: Homo sapiens
manufacture_protocol: 1. Selected BAC clones were grown in 200uL of 2xYT medium supplemented with suitable antibiotic (either cloramphenicol or kanamycin) in a 96 deep-well plate, for 24 hours at 37
  2. with gentle shaking (320rpm). We added 40 uL of glicerol 87% (v/v) to the grown bacterial culture, shaked to ensure mixing and preserved at -80ºC.
  3. We grew new plates from the glicerinated ones before extracting DNA from clones using a Millipore kit (Montage BAC96 Miniprep Kit, P36392, Rev. 08/01). BAC-end sequences
  4. were obtained from the purified product.
  5. The extracted DNA was amplified by DOP-PCR (R1 & R2, Fiegler et al., 2003, Genes, Chromosomes and Cancer, 36, 361-374).
  6. Amplified products were purified using the Kit Montage PCR96 Plates, Millipore ref. MSNU03010.
  7. Quantitation of the purified product was done with PicoGreen dsDNA Quantitation Kit (Molecular Probes, P-7589).
  8. DNA was subjected to evaporation using a speed-vac, prior to adjust final concentration at 400ng/uL in spotting buffer (DMSO 50% v/v in water). DNA was then denatured at 99C
  9. for 2 minutes. 8uL were placed on a spotting plate before depositing it on the surface of UltraGAPS Slides (Corning, 40015), using a VersArray Chip Writer spotter (BioRad).