title: EBT UA_KUL Daphnia magna unique_1.9K microarray
status: Public on Feb 01 2012
contact.city: Antwerpen
contact.first: Nathalie
contact.last: Dom
contact.email: nathalie.dom@ua.ac.be
contact.address: Groenenborgerlaan 171
contact.department: Biology
contact.country: Belgium
contact.institute: University of Antwerp
contact.phone: +3232653532
organism: Daphnia magna
manufacture_protocol: This microarray is composed of 3 cDNA libraries which are the result of 3 SSH procedures. One SSH was performed with adult vs juvenile gene pools, one with ecdyson exposed vs control organisms and one with starved vs well fed organisms (Soetaert et al., 2006; 2007). The unique sequences selected via those SSHs were supplemented with additional set of unique gene fragments related selected through 3 other SSHs related to other stress factors (fish, Pasteuria ramosa and carbaryl stress) (Orsine et al., 2011).
  In general, the differentially expressed genes were ligated in a TA-vector system using the pGEM-T easy vector (Promega, USA). Escherichia coli competent cells (JM109, Promega) were transformed through heat shock. Clones were amplified with vector specific primers (M13 forward and M13 reverse) and purified by an exosap reaction. In preparation for spotting, cDNA-inserts of the clones were, amplified using adaptor specific primers (nested primers, Clontech), purified by Montage PCR Plate (Millipore, USA) and loaded into 384-well plates (Genetix, UK) in 50% dimethylsulfoxide at ~150 ng/µl. A Qarray Mini Robot (Genetix) with 16-solid pin-head was used to spot each clone in triplicate onto aminosilane coated glass slides (Generoma microarray slides; Asper Biotech, Estonia).