title: Shanghai Biochip Staph epidermidis 116 cDNA array
status: Public on Mar 08 2010
contact.city: Shanghai
contact.first: Tao
contact.last: Zhu
contact.email: 071101036@fudan.edu.cn
contact.address: 138, Dongan Road
contact.country: China
contact.institute: Fudan university
organism: Staphylococcus epidermidis
manufacture_protocol: 1. Non-homologous DNA fragments of ~500 bp in length were PCR-amplified in 100 µl reactions with the specific primer, purified by an isopropanol precipitation and resuspended in water. The DNA concentration was determined by measuring the fluorescence signal obtained in the presence of the dye Hoechst-33258. Purity of the fragments was checked by agarose gel electrophoresis.
  2. Poly-L-lysine-coated glass slides of 75 × 25 mm were prepared as described (http://cmgm.stanford.edu/pbrown/MGuide). Slides with aminosilane surface (CMT-GAPS™) were purchased from Corning (Corning, USA). The DNA spotting solution was adjusted to either 45 mM sodium citrate pH 7.0, 450 mM NaCl (3× SSC) or the same composition supplemented with 1.5 M betaine (N,N,N-trimethylglycine; Sigma, Germany).
  3. DNA spotting was done with an SDDC-2 DNA Micro-Arrayer from Engineering Services Inc. (Toronto, Canada). A single SMP3 pin (TeleChem International Inc., Sunnyvale, USA) was used to avoid differences between pins. The DNA samples were printed in quadruplicate at a 200 µm centre-to-centre spacing. Slides were left at room temperature overnight and then heat-treated on a metal block at 80°C for 5 s.
  4. The DNA was crosslinked to the support by UV irradiation with a total energy of 60 mJ in a Hoefer UV-crosslinker (Amersham Pharmacia Biotech, Freiburg, Germany).
  5. For the blocking process, 1 g succinic anhydride (Fluka, Deisendorf, Germany) was freshly dissolved in 200 ml anhydrous 1,2-dichloroethane (DCE; Fluka). To this solution, 2.5 ml of N-methylimidazol (Fluka) was added and immediately poured into the slide chamber. Incubation was for 1 h, placed on an orbital shaker for slight agitation.
  6. Subsequently, the slides were briefly washed in 200 ml of fresh DCE and incubated in boiling water for 2 min for DNA denaturation. After a brief rinse in 95% ethanol, they were left to dry at room temperature.