title: EBT UA Daphnia magna 1.2K version 1
status: Public on Dec 31 2009
contact.city: Antwerp
contact.first: Tine
contact.last: Vandenbrouck
contact.email: tine.vandenbrouck@ua.ac.be
contact.address: Groenenborgerlaan 171
contact.department: Department of Biology
contact.country: Belgium
contact.institute: University of Antwerp
contact.phone: +32 3 265 33 50
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). 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). In addition, a set of artificial genes control genes (Lucidea Universal Scorecard, Amersham Biosciences, UK) was spotted in 20 replicates spread over each array: 10 calibration controls, 8 ratio controls and 2 negative controls. After rehydration and drying, the slides were fixated by UV-radiation at 300mJ (UV Stratalinker 2400; Stratagene, USA).