title: GPC Biotech Bacillus subtilis 4K
status: Public on Mar 01 2008
contact.city: Martinsried
contact.first: Christoph
contact.last: Schaab
contact.email: cswork@web.de
contact.address: Fraunhoferstrasse 20
contact.country: Germany
contact.web_link: http://www.gpc-biotech.com
contact.institute: GPC Biotech AG
organism: Bacillus subtilis
description: The array contains 4205 oligonucleotide probes representing the complete Bacillus subtilis genome. The probes were spotted in duplicates onto nylon membranes.
manufacture_protocol: DNA fragments for open reading frames (ORFs) were computationally selected within the first third (proximal to the 5' end) of the coding region. Each fragment was checked for its uniqueness; up to 80% similarity with any other sequence on the genome was the maximum allowed. No DNA stretch greater than 24 nucleotides was identical to any other such stretch (with the exception of ORFs shorter than 140 bp, which were used in their entirety). Two or three fragments were generated for ORFs longer than 3,000 bp. Each fragment was prepared by annealing two 80-base oligonucleotides designed to overlap at their 3' ends to form a 20-bp duplex. An extension reaction was then performed to yield a full-length product of 140 bp. Briefly, each pair of
  oligonucleotides was annealed in 20 mM Tris (pH 8.8)–10 mM KCl–10 mM
  (NH4)2SO4 and extended overnight at 65°C in the presence of 1.5 mM (each) deoxynucleoside triphosphate, 10 mM MgSO4, 5x Q solution (Qiagen), and 8 U of Bst DNA polymerase (New England Biolabs, Beverly, Mass.).
  Arrays were spotted using a robotic system from KAYBEE (UK) on a nylon membrane Biodyne B (Pall, UK). The spotting head contained 384 solid flat-bottom pins of 250 µm in diameter constructed in a format complementary to the polystyrene 384-well plates. The size of each spot, estimated by spotting ink and fluorescently labelled DNA, was ~350 µm and the distance between each spot was 833 µm.