title: Daphnia magna cDNA microarray ver. 2
status: Public on Jun 09 2008
contact.city: Boston
contact.first: Helen
contact.middle: C
contact.last: Poynton
contact.email: helen.poynton@umb.edu
contact.state: MA
contact.address: 100 Morrissey Blvd.
contact.department: School for the Environment
contact.country: USA
contact.institute: UMass Boston
contact.phone: 617-287-7323
organism: Daphnia magna
manufacture_protocol: The 5000 randomly selected cDNA clones from the Daphnia Genome Consortium (DGC) library were PCR amplified from the pDNR-LIB vector using the following primers: forward: AGTCGACGGTACCGGACATA and reverse: GCCAAACGAATGGTCTAGAAA.  An additional 2681 cDNAs were constructed at the US Army Engineer Research and Development Center in Vicksburg, MS.  Daphnia magna were exposed to twelve model toxicants for 24 h at the 1/10 LC50 and RNA was isolated using standard protocols for Trizoll (Invitrogen).  Forward and reverse subtracted libraries were created using the PCR-Select cDNA subtractive kit (Clontech).   cDNAs were cloned into the pCR4 vector using the pCR4-TOPO TA cloning Kit from Invitrogen.  PCR products were purified by ethanol precipitation and resuspended in distilled water. cDNA clones were printed onto lysine-coated glass slides (Cheung et al., 1999) by the UCB Nutritional Sciences and Toxicology genomics facility.
  Cheung, V. G., Morley, M., Aguilar, F., Massimi, A., Kucherlapati, R. & Childs, G. (1999) Making and reading microarrays. Nat Genet. 21, 15-19.