title: Pfu gamma v1
status: Public on Jul 02 2005
contact.city: College Park
contact.first: Jocelyne
contact.last: DiRuggiero
contact.email: jd92@umail.umd.edu
contact.state: MD
contact.address: 3221 H.J. Patterson Hall
contact.department: Cell Biology and Molecular Genetics
contact.country: USA
contact.institute: University of Maryland
contact.phone: 301-405-4598
organism: Pyrococcus furiosus
description: P. furiosus full-genome microarray numbered 2065 protein encoding genes, and 108 RNA genes (transfer RNAs, ribosomal RNAs, small RNAs). In addition, all "intergenic" regions of 60 nucleotides or larger were represented on the array (602 total).  DNA fragments for spotting were produced by PCR. The array was constructed by designing oligonucleotide primers to span the most unique portions of each gene, averaging 350 nucleotides in length.  A custom Perl program was written to select 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. 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 poly-L-lysine coated glass slides twice, each time by a different print pin (print plates were rotated 180 degrees for second spotting) using a 16-pins ArrayMaker Version 1.0 (http://cmgm.stanford.edu/pbrown/mguide/index.html).
  Keywords = archaea
  Keywords = gamma
  Keywords = DNA repair
  Keywords = extremophile