title: 11K Ovita Ovine Array print 67
status: Public on Sep 06 2006
contact.city: Dunedin
contact.first: Orla
contact.middle: Mary
contact.last: Keane
contact.email: orla.keane@agresearch.co.nz
contact.state: Otago
contact.address: 710 Cumberland St
contact.department: Animal Genomics
contact.country: New Zealand
contact.institute: AgResearch
contact.phone: 00 64 3 4797659
organism: Ovis aries
description: This array contains spotted PCR products from an ovine EST collection. Included are housekeeping genes, water blanks (negative PCR) and no DNA blanks (to estimate carryover)
manufacture_protocol: Ovine EST inserts were amplified in 96 well plates using the universal primers SP6 and T7 in 50 ul reactions (1.5 mM MgCl2, 2 mM each dNTP, 45 pmol primers and 2.5 units AB Red Hot Taq polymerase). The reactions were denatured at 94C for 3 minutes then cycled 36 times at 94C for 45 sec, 55C for 45 sec and 72C for 60 sec. Finally products were extended at 72C for 5 min. The PCR products were verified by agarose gel electrophoresis, precipitated and resuspended in printing solution. An ovine microarray consisting of 11,520 spots was printed onto poly-L-lysine coated glass slides using an ESI array robot with 32 split pinheads depositing 0.6 nl with a 100 um spot size. After printing the slides were UV irradiated to cross-link the DNA to the polylysine coating.