title: SA-DEV-VS1
status: Public on Jan 26 2007
contact.city: Heraklion
contact.first: Elena
contact.middle: Athina
contact.last: Sarropoulou
contact.email: sarris@imbc.gr
contact.department: Marine Biology and Genetics
contact.country: Greece
contact.institute: HCMR
contact.phone: +302810227819
organism: Sparus aurata
description: For the microarray production a cDNA library of mixed embryos/larvae of gilthead sea bream, Sparus aurata (Sarropoulou et al., in press) was used. A total of 10,176 clones were arrayed into 384 square well plates. In order to amplify the inserts culture PCR was performed as followed. The total volume of one PCR reaction was 50 µl, containing 43.5 µl reaction mix (2 µl 10x PCR Puffer, 0.04 µl 100 mM of each dATP, dCTP, dGTP and dTTP, 12.12 µl dH20), 1.5 µl of T7 and 1.5 µl of T3 primer (20 µM), 1 µl Taq polymerase (5U/µl) and 2.5 µl of culture. The PCR program utilized consisted of 1 step of 94ºC for 1 min 20 s, followed by 35 cycles of 94ºC for 1 min, 50ºC for 1 min, 72ºC for 1 min 30 s; the program terminated with a final step of 72ºC for 5min. Each reaction was performed twice; PCR products were combined, separated by agarose gel (2%) electrophoresis and visualized by transillumination in the presence of ethidium bromide (EtBr) in order to check insert size. Purification of PCR products was perfomed via the GFX-96TM Polymerase Chain Reaction (PCR) Purification Kit (Amersham Pharmacia) according to the manufacturer’s instructions. The purified products were then dried down by applying vacuum, re-eluted in 3x SSC and 1.5 M betaine to a volume of 10 ml and transferred to 384 well plates.
  The spotting was performed with the OmniGrid 300 robotic system configured with 48 pins. Each glass slide contains 10,176 PCR amplified inserts in duplicate. The spotted glass slides were baked at 80ºC for 2 h in order to cross-link the DNA onto the glass slide. The slides were then incubated in blocking buffer at 50°C for 30 min, washed twice with Millipore water at room temperature for 2 min and dried by spinning in an Eppendorf 5810R centrifuge for 7 min at 800 rpm.
  
  