title: CCG-UNAM Rhizobium etli CFN42 6051 v1.0
status: Public on Mar 10 2010
contact.city: Cuernavaca
contact.first: Sergio
contact.middle: Manuel
contact.last: Encarnación
contact.email: encarnac@ccg.unam.mx, esalazar@ccg.unam.mx
contact.state: Morelos
contact.address: Av. Universidad S/N
contact.department: Centro de Ciencias Genomicas
contact.country: Mexico
contact.institute: Universidad Nacional Autonoma de México
contact.phone: 777 3291899
organism: Rhizobium etli CFN 42
description: Whole genome expression microarray for Rhizobium etli CFN42, representing the all open reading frames (ORF) of the chromosome and all 6 plasmids (NC_007761, NC007762, NC_007763, NC_007764, NC_004041, NC_007765, NC_007766), included repeat regions, 70mers, Ctrl probes replicated 8 times, negative control probes replicated 16 times.
manufacture_protocol: The oligonucleotide Rhizobium etli CFN42 70-mers, were designed by using several ad hoc programs written in PERL and other software as follows: A first program read the DNA sequences corresponding to each of the annotated genes of the R. etli CFN42 genome. If the size of the gene permitted it, the program then cut 50 bases from both the 5´ and the 3´ ends. After that, the program scanned, base by base, each possible 70-mer from the sequence, ensuring that there was no single base repeat longer than 7 bp, and a Melting point of 80 ±5 °C (the formula used for calculating TM was: Tm = 81.5 + 16.6 x log[Na+] + 41 x (G + C)/length - 500/length, with Na+ = 0.1 M; length = 70). The resulting candidate oligonucleotides were compared against the complete DNA genome sequence using NCBI’s BLASTALL (blastall –p blastn) with a maximum e-value of 1.0 (-e 1.0), and no masking of low information segments (-F F). The BLAST results were used to ensure that no candidate oligonucleotide would cross-hybridize in the microarray. We used the program PALINDROME from the EMBOSS software suite to find hairpins and filter out any candidate oligonucleotides with potential hairpins longer than 9 bases. Our programs also ensured that no oligonucleotide would have more than 20 bases in common to any other coding sequence. A control was also designed with 4 positive controls and 12 negative controls using the Drosophila melanogaster genome. All oligonucleotides were commercially synthesized without modification by MWG-BIOTECH (Ebersberg, Germany), prepared at 50 uM in Micro Spotting solution (ArrayIt Brand Products) and spotted by duplicate onto SuperAmine coated slides (25 × 75 mm; TeleChem International, INC) using the Virtek Chiprender Professional Arrayer at 20°C and 60% humidity the microarray facility at Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Finally, the slide was fixed at 80 °C for 4 h.