title: Vulpe Lab UC-Berkeley Fathead Minnow v1.0
status: Public on Apr 07 2008
contact.city: Miami
contact.first: Edward
contact.last: Mager
contact.email: emager@rsmas.miami.edu
contact.state: FL
contact.address: 4600 Rickenbacker Cswy.
contact.department: MBF
contact.country: USA
contact.institute: University of Miami, RSMAS
organism: Pimephales promelas
manufacture_protocol: 1.	A fathead minnow cDNA library was constructed in lambda TriplEx2 using the SMART cDNA kit (Clonetech, Mountain View, CA) using a pool of RNA extracted from unexposed fathead minnow eggs, and fish of different ages (less than 5, 5-10, 10-60, 60-150 and more than 150 day-old).
  2.	The library was amplified, plated and 5,000 randomly selected plaques were grown in 96 well plates to produce phage supernatant.
  3.	1 µl of phage supernatant was used as template to PCR amplify the cDNA inserts using two custom made oligonucleotides encompassing the SfIA cloning sites (SfiA: GGCCATTACGGCCGGG and SfiB: CGAGAGGCGGCCGACAT). PCR reactions were carried out in 96 well plates in a 50 µl volume using an amplification program consisting of 3 min at 94°C, followed by 30 cycles of 30 sec at 94°C, 1 min at 65°C, 2 min at 72°C.
  4.	A sample of a subset of the reactions from each 96 well plate were analyzed on agarose gel.
  5.	All PCR reactions were cleaned by ethanol precipitation, followed by a washing with 70% ethanol and dissolved overnight in 50 µl of  3xSSC under gentle shaking.
  6.	PCR reactions were arrayed in Corning polypropylene 384 well plates using a Packard Bioscience Multiprobe IIex liquid handler (Packard .
  7.	Printing was done on polyamine coated slides using a custom made printer equipped with capillary print tips. Each cDNA was spotted twice.