title: Blue-Chip bovine preimplantion embryo specific cDNA array produced by Sirrard et al. 2005
status: Public on Mar 25 2010
contact.city: Bonn
contact.first: Dessie
contact.last: Salilew-Wondim
contact.email: dsalilew@uni-bonn.de
contact.state: NRW
contact.address: Endenicher Allee 15
contact.department: Animal breeding and husbandry group
contact.country: Germany
contact.institute: University of Bonn
organism: Bos taurus
description: Submitter was unable to provide further Platform annotations
manufacture_protocol: 1. Total RNA was extracted from oocyte, blastocyst and somatic tissue using absolutely RNA Microprep Kit
  2. Total RNA was then reverse transcribed  and amplified using the BD SMART PCR SYNTHESIS KIT
  3. Amplified double stranded cDNA was RsaI digested and purified using the QIAquick PCR Purification Kit
  4. Different SSH experiments were then performed for oocytes, blastocyst and somatic tissue
  5. Differentially expressed cDNA coming from SSH experiments using oocytes as a tester and blastocyst as a driver, and Blastocyst as a the tester and somatic tissue as a driver were cloned into the pGEM-T Vector systems or into TOPO cloning 5-min PCR Cloning KIt
  6. Vectors were then transformed into One Shot MAX efficiency® DH5α-T1R competent cells
  7. Transformed bacterial were selected using blue white screening
  8. Between 480 and 1152 white colonies per SSH experiment were then picked and resuspended in LB+ampicilin
  9. PCR was performed  from bacterial suspension using HotMaster Taq DNA polymerase
  10. The PCR product was purified using Unfilter ® 384-well purification plate, speedvac evaporated and resuspended in 3X standard saline citrate
  11. cDNA were spotted on GAPS II glas slide using versearray chipwritter proRobot.
  12. Slides were then cross-linked with ultravuiolet (UV) rays.