title: EBT_UA_Daphnia magna_2.3K_version2
status: Public on Jul 23 2010
contact.city: Gent
contact.first: Michiel
contact.last: Vandegehuchte
contact.email: Michiel.Vandegehuchte@UGent.be
contact.address: Jozef Plateaustraat 22
contact.department: Applied ecology and environmental biology
contact.country: Belgium
contact.institute: Ghent University (UGent)
contact.phone: +3292643707
organism: Daphnia magna
manufacture_protocol: Three D. magna cDNA libraries enriched with genes related to energy metabolism, molting and life stage specific processes have been developed by Soetaert et al. (2006 and 2007) using the suppression subtractive hybridization technique. 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. Next to these cDNA libraries, two extra cDNA fragments, corresponding to expressed sequence  tags (ESTs) from  genes  that  are  reported  to  be  sensitive to Zn, were spotted on the array: ESTs with homology to (1) ferritin (AJ292556) and (2) retinol dehydratase (DV437801) gene fragments (Poynton et al., 2007). Finally, also two ESTs with homology to putative MTs (metallothioneins) (DV437799 and DV437826) were spotted. These sequences were PCR-amplified from a D. magna cDNA sample, checked by electrophoresis, purified by Montage PCR Plate (Millipore, USA) and loaded into 384-well plates (Genetix, UK) in 50% dimethylsulfoxide at a final concentration of 50–75 ng/µL. The isolated cDNA clones from the cDNA libraries were PCR-amplified from the pGEM-T easy vector using adaptor-specific primers (nested primers, Clontech) after which the same control, purification and loading steps were performed. The cDNA fragments (average length of 415 bp) were spotted in triplicate on aminosilane-coated glass slides (Generoma microarray slides, Asper Biotech, Estonia). A set of artificial control genes (Lucidea Universal Scorecard, Amersham Biosciences, UK) was also spotted. After rehydration and drying, the  cDNA  fragments  were  cross-linked  to  the  slides  using  UV-radiation at 300 mJ (UV Stratalinker 2400, Stratagene, USA).