title: Pyrobaculum aerophilum full-genome spotted PCR fragment microarray
status: Public on Apr 23 2008
contact.city: Santa Cruz
contact.first: Todd
contact.middle: Michael
contact.last: Lowe
contact.email: tmjlowe@ucsc.edu
contact.state: CA
contact.address: 1156 High Street / MS: SOE-2
contact.department: Biomolecular Engineering
contact.country: USA
contact.institute: University of California, Santa Cruz
contact.phone: 4082064112
organism: Pyrobaculum aerophilum str. IM2
description: Arrays were printed by double-tapping 16-pins on each individual slide, producing a 32 blocks of 24 rows by 24 columns of spots.
manufacture_protocol: The Pyrobaculum aerophilum (Pae) microarray was designed to assay the expression levels of all predicted protein coding and RNA genes. These number 2350 "high confidence" protein gene predictions, 250 "low confidence" gene predictions (AORFs), and 102 RNA genes (transfer RNAs, ribosomal RNAs, small RNAs).  The array was constructed by designing oligonucleotide primers to span the most unique portions of each protein coding gene, averaging 350 nucleotides in length.  A custom Perl program was written to pick primers with the most desirable thermodynamic conditions possible for each gene using the Whitehead Institute's Primer3 program (version 3.09) as its core analysis engine.  In addition, all "intergenic" regions of 60 nucleotides or larger are represented on the array. Primers for intergenic regions and RNAs were designed to cover the entire target region. The polymerase chain reaction was performed on 100 µl reactions with 1µM of each primer, 2mM MgCl2, 1.0 mM dNTPs, 1.75 U Amplitaq DNA polymerase, 1x Amplitaq buffer, and 20ng genomic DNA for 30 cycles (94°C 30 sec, 50°C 1 min., 72°C 2 min.) to generate DNA fragments. All PCR products were checked by agarose gel electrophoresis, and failures were repeated or new primers designed. Each PCR amplicon was spotted on Corning UltraGAP II glass slides in duplicate by two separate print pins, so that each measured feature is represented at four separate locations on the microarray.  Arrays were printed using a "bullet train" robotic spotter constructed at UC Santa Cruz using the MGuide Version 2.0 manual (http://cmgm.stanford.edu/pbrown/mguide/index.html).   Arrays were printed with 16-pins, using the ver. 1.8.8 ArrayMaker software.