title: OSU Tick salivary gland v.1
status: Public on May 13 2006
contact.city: Stillwater
contact.first: Richard
contact.middle: C.
contact.last: Essenberg
contact.email: richard.essenberg@okstate.edu
contact.state: OK
contact.address: 246 NRC
contact.department: Department of Biochemistry and Molecular Biology
contact.country: USA
contact.institute: Oklahoma State University
contact.phone: 405 744-6189
organism: Amblyomma americanum
manufacture_protocol: Plasmid DNA templates were chosen from three EST libraries. About 10 ng each of 1152 purified plasmid DNAs chosen from the three libraries were PCR amplified in 100 μl total volume using Hot Start Platinum® Taq DNA polymerase (Invitrogen, CA) and the 5` and 3` sequencing primers appropriate to the vector. The amplification was done according to the PCR program; 94°C for 2 min, 26 cycles of (94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min), followed by a final extension step at 72°C for 20 min.  The PCR samples were purified using Multiscreen™ filter plates (Millipore, MA) according to the manufacturer and random purified PCR products were run on 1% agarose gel to estimate the yield. The purified PCR products were transferred to 384 well microarray printing plates (Genetix, UK), dried at 42°C for 6 hours and reconstituted with 3X SSC printing solution to a final concentration of ~150 ng/μl. Eight ArrayControl™ control PCR spots (Ambion, TX) were reconstituted with printing solution to the final concentration of 160 ng/μl. The printing was performed on ArrayIt™ SuperAmine substrates (TeleChem, CA) using the Cartesian pix 5500 (Genomic Solutions, MI) microarrayer in 56% relative humidity. Four pins were used for spotting, arranged in a row.  Each PCR product was printed four times on each slide. After drying, the slides were baked at 85°C for two hours and stored at room temperature in a desiccator. Prior to hybridizations, slides were washed with 1% SDS then boiled for two minutes in sterile water, snap-cooled in ice-cold 95% ethanol and then dried by 10 sec centrifugation in ArrayIt™ slide centrifuge (TeleChem, CA). The quality of the printed slides was investigated by Syto61 staining.