title: Maize oligonucleotide array 1.5K version
status: Public on Apr 03 2014
contact.city: Hamburg
contact.first: Stefan
contact.last: Scholten
contact.email: stefan.scholten@uni-hamburg.de
contact.state: Hamburg
contact.address: Ohnhorststrasse 18
contact.department: Biocenter Klein Flottbek
contact.country: Germany
contact.institute: University of Hamburg
contact.phone: 0049 40 42816329
organism: Zea mays
manufacture_protocol: PRINTING OF THE OLIGONUCLEOTIDE MICROARRAYS
  The oligonucleotides were synthesized by Ocimum Biosolutions (Ijsselstein, Netherlands) and printed on poly-L-lysine coated glass slides with a Microgrid II printer (BioRobotics, Boston, USA).
  1. Add 15 uL of 3x SSC to each well of the 300 µM oligonucleotide set to make a 20 μM oligonucleotide solution.
  2. Transfer onto an orbital shaker for 1 h at RT at 100 rpm. The oligonucleotides are now ready for printing.
  3. The maize oligonucleotide microarrays are printed on poly-L-lysine coated glass slides (Menzel, Thermo Fisher Scientific) using microspot 2500 pins.
  4. Printing conditions:
  Microarray printer (Microgrid II printer, BioRobotics, Boston, USA). 55% relative humidity. We used microspot 2500 pins and spotted all oligos and controls (used negative controls: H300010599, H300013976, H300014151, H300016837; positive controls: Control_oligo-7, Control_oligo-12, MZ00023408, MZ00023618, MZ00023663 in two replicates.
  IMMOBILIZATION OF THE OLIGONUCLEOTIDE ARRAY ELEMENTS
  1. Rehydrate the microarray slide over 56 °C water for 2-3 seconds; snap-dry on a 65 °C heating block for 5 seconds. Repeat this step 3 times, examining the slides under a dissection microscope to verify complete re-hydration of all the spots. Crosslink the oligonucleotides to the slide surface by exposing the DNA-side of the microarray to UV irradiation. We employ a Stratalinker set at an energy level of 60 mJ. After crosslinking slides were once again dried on a 100 °C heating block.
  2. Blocking of the slides was performed according to Diehl et al. (Nucleic Acids Research 2001, 29(7): e38). Therefore slides were incubated for 1 h in blocking solution (1 g succinic anhydride, 200 ml 1,2-Dichlorethane, 2.5 ml 1-Methylimidazole), then dipped twice in 1,2-Dichlorethane. At the end slides were placed for 2 min in boiling water and then briefly transferred to 98 % ethanol.
  3. Air dry slides under a clean bench.
  4. Store the slides in a light-proof box in a vacuum.