title: Molecular Immunogenetics Group FMRP-USP Human cDNA 4.6K
status: Public on Feb 28 2010
contact.city: Rio de Janeiro
contact.first: Marcelo
contact.last: Ribeiro-Alves
contact.email: mribalves@gmail.com
contact.state: RJ
contact.address: Avenida Brasil 4365. Manguinhos.
contact.department: Centro de Desenvolvimento Tecnológico em Saúde
contact.country: Brazil
contact.institute: Fundação Oswaldo Cruz
organism: Homo sapiens
manufacture_protocol: 1. The Molecular Immunogenectis Group keep a human cDNA library (MTB IMAGE) with 4,500 clones from IMAGE Consortion. The clones are  0.75–1.0 kb from expressed sequence tags (ESTs) sequenced and identified with Clone ID. Each clone is preserved in plasmids in 384 wells microplates at -80°C.
  2. The clones were transferred for 384 well microplates with 2x LB medium and ampicilin and incubated during 16 hours.
  3. After incubation 8mL of culture were transferred to new 384 microplate with 72mL of water and incubated 10 minutes in 95°C.
  4. The plates were centrifugate 4000g during 5 minutes and 10mL of supernant was used for PCR  with consensus primers for the library: primer LBP 1S (5´TGTGGATTGTGAGCGGATAA3´) e primer 1AS (5´GGGTTGAATTAGCGGAACG3´).
  5. The PCR program was 5 min a 94°C, followed by 30 cycles (94°C-30 seg.; 55°C-30 seg., 72°C-2 min.), and a final step of 7 min. at 72°C and 5 min. at 4°C.
  6. The quality of PCR product was verified by eletrophoresis in 1% agarose gel.
  7. The PCR products were purified with sodium acetate 3M pH4.8 and absolut ethanol for 16 hours at -20°C.
  8. Purified PCR products and D reagent (GE Healthcare) in same volume were transferred for 384 microplates using Array Spotter III (Amersham Molecular Dynamics).
  9. The microarray were constructed on silane-coated UltraGAPS slides (Corning ® UltraGAps 40015) using a Generation III array spotter (Amersham Biosciences).
  10. PCR products were deposited by a set of 12 pens that deposit 0,9nL of each sample by capillarity.
  11. After print each sample group the 12 pens were washed in purified water, absolut ethanol, 0.2M KOH solution and water again. And dried with liquid nitrogen before print other samples.
  12. After the print run is completed, allowed slides to dry and fixed the DNA by cross-linking by ultraviolet radiation to 500mJ of energy (Hoefer UV Crosslinker).