title: Eurogentec Bacillus subtilis microarray
status: Public on Jan 01 2008
contact.city: Vancouver
contact.first: Heather
contact.last: Maughan
contact.email: maughan@zoology.ubc.ca
contact.state: BC
contact.address: Life Sciences Centre
contact.department: Zoology
contact.country: Canada
contact.institute: University of British Columbia
contact.phone: 604 822-6323
organism: Bacillus subtilis
manufacture_protocol: Product description
  
  The B. subtilis genome contains 4096 putative open reading frames (ORFs). 8192 primers were designed and synthesized to amplify 4096 ORFs in the B. subtilis genome. The primers were designed to amplify each ORF beginning at the start codon and ending at the stop codon. Each primer contains 15-base non-variable sequence at the 5’-end followed by 18 to 22 bases of ORF-specific sequence. A high fidelity PCR system was used to obtain PCR products. The PCR reactions were performed in two rounds using 96-well plates in a 100 ml reaction volume. The first round of PCR reaction (primary PCR) was done using 10 ng of DNA genomic as template and the specific part of the oligo. The second round of PCR reactions (secondary PCR) was performed in the presence of 5 ul of 40-fold diluted primary PCR products and the C12-NH2 modified universal primers. The entire primary and the secondary PCR reactions were controlled on standard gel electrophoresis for yield and for the expected size. Using these two-step methods we have successfully amplified up to 96 % of 4096 B. subtilis ORFs. The 5’-aminated PCR products were purified using Millipore 96-well purification kit, concentrated to 200 to 500 ng per microliter and then covalently immobilization onto aldehyde coated glass slide through a well-known Schiff base reaction.
  
  Spotting design
  
  B. subtilis MicroArrays are manufactured by printing PCR amplicons suspended in optimized spotting buffer for high coupling efficiency of DNA to the most consistent aldehyde coated glass slides. This spotting chemistry provides us the highest hybridization intensities, which is a consequence of a better binding of the probe to the slides and their accessibility to the cDNA targets. We use ChipWriter™ Pro (Virtek) which is a high precision dispensing robot to spot the PCR products onto aldehyde glass slides. The robot is designed to collect 100 nl of DNA solution and to deposit 0.6 nl per spot. For B. subtilis MicroArray, we use simultaneously 16 pins to place the spots in area of 1.8 x 1.8 cm. In order to generate high quality spots, the printing procedure is performed under a tight controlled humidity and temperature environment. This allows having the complete control on the spot morphology, the spot diameter and uniformity. This configuration of the robot generates 16 blocks, each containing 22 x 24 dots with 180 microns spacing center to center with a spot diameter of 100 microns.