title: Bovine Total Leukocyte cDNA microarray: Fifth generation microarray
status: Public on Oct 16 2007
contact.city: Dublin
contact.first: David
contact.middle: E
contact.last: MacHugh
contact.email: david.machugh@ucd.ie
contact.address: University College Dublin, Belfield
contact.department: College of Agriculture, Food Science and Veterinary Medicine
contact.country: Ireland
contact.institute: University College Dublin
contact.phone: +353-1-7166256
organism: Bos taurus
manufacture_protocol: Clone inserts from the BOTL library were amplified by inoculating bacteria directly into PCR master mix (20 μM each dNTP, 0.2 μM M13-forward primer, 0.2 μM M13-Reverse primer, 200mM Tris-HCl (pH 8.4), 500mM KCl, and 2.0 units of Taq DNA polymerase) in 96-well plates. Following a pre-heat step of 95°C for 10 min to disrupt the bacteria, PCR was performed in a PE 9700 Thermocycler (Perkin Elmer Corp., Palo Alto, CA) using the following conditions: 95°C for 30 sec; 55°C for 30 sec and 72°C for 1 min. Based on agarose gel electrophoresis of purified amplicons, our success rate of clone insert amplification with this protocol has typically been >90%. Insert amplicons were purified in 96-well format using Millipore filter plates (Millipore Corp., Bedford, MA) filled with Sephadex G-50 (Amersham Pharmacia Biotech.). Final insert amplicons were resuspended in 17 μl of 50% DMSO for low-density (3000 to 4000 amplicons per slide) spotting on microarrays. Approximately 1 μl of each purified insert amplicon was separated on a high-volume 1.2% agarose gel to ensure that each clone was represented on the final microarrays. This check also allowed final adjustment for differences in amplicon concentration just prior to microarray source plate set up. Finally, a total of 5 μl of each purified amplicon was transferred to 384-well microarray source plates.
  Microarrays were spotted using a GeneTAC G3 arraying robot (Genomic Solutions, Inc., Ann Arbor, MI) equipped with a 48-pin head, with each pin having a nominal end diameter of 200 microns. In 50% DMSO (low-density arrays), this arrangement yields spots of approximately 250-350 microns. The pin configuration of the G3 yields microarrays consisting of 48 “patchs” of spots. (81 spots per patch).