title: Zebrafish 8K cDNA array TKH
status: Public on Jan 01 2013
contact.city: Clemson
contact.first: Qing
contact.last: Liu
contact.email: liuqingcell@gmail.com
contact.state: SC
contact.address: Jordan Hall
contact.department: Biological Sciences
contact.country: USA
contact.institute: Clemson University
organism: Danio rerio
manufacture_protocol: 1. The normalized zebrafish cDNA library were purchased from RZPD (RZPD library MPMGp609)
  2. Plates 1-20 (7680 clones) and an additional 40 clones from various resource were used as a source of probe DNA for the microarray
  3. The partial library was replicated into 384 deep-well plateds and cultures were grown in 90 ul SuperBroth supplemented with 100 mg/ml ampicilli, incubated with shaking fro 14-15 hours at 37 °C.
  4. PCR of the clones were done directed from the E.coli culture using 384 slot pin replicator tool (VP Scientific),0.5 ml bacterial culture using 0.26 uM vector primer in a 20 ml reaction consisting of 1XMg-free PCR buffer (ROche), 1.5 mM MgCl2,  0.2 mM each dNTP, 1 M Betaine and 0.5 U/rnx Taq polymerase (Roche, Indianapolis, IN).The 384 reactions were incubated at 94°C for 5 min and 19 cycles of 94°C for 1 min, 60°C(decreasing 0.5°C for each cycle) for 1 min  and 72°C for 1.5 min, followed by 15 cycles of 94°C for 1 min,55°C for 1 min  and 72°C for 1.5 min, and terminated by a 7 min extemsion at 72°C.
  5: PCR products were analyzed for quality by 1% agarose gel electrophoresis analysis. Products were purified using 384 PCR purification plates (Millipore, Bedford, MA) and Sciclone ALH robotics..
  6. Microarrays (two per slide) were printed at a density of 8064 probes/array (4X4X21X24) on poly-L-lysine slides using a GeneMachines Omni Grid printer with sixteen Telechem International SMP3 pins.