title: Stanford University/Stevens Lab Paracoccidioides brasiliensis 12K v1.0
status: Public on May 08 2009
contact.city: San Jose
contact.first: Jomar
contact.middle: Patrício
contact.last: Monteiro
contact.email: jomar@stanford.edu
contact.state: CA
contact.address: 2260 Clove Drive
contact.department: Division of Infectious Diseases and Geographic Medicine
contact.country: USA
contact.institute: Stanford University
organism: Paracoccidioides brasiliensis
manufacture_protocol: Library construction:
  	Genomic DNA was sheared hydrodynamically into ~1.5 Kb fragments using a HydroShear® apparatus (Genomic Solutions, Ann Arbor, MI) as previously described (Oefner et al., 1996; Thorstenson et al., 1998).  Adequacy of DNA shearing was assessed by 1.5% agarose gel electrophoresis and visualized by UV after ethidium bromide staining.  Sheared DNA was used as inserts for the genomic library.
  	The genomic library was assembled using the λ ZAP II phage as vector (λ ZAP II Predigested EcoRI/CIAP-treated Vector Kit, Stratagene, La Jolla, CA) and a modification of the linker-adaptor strategy described by Thorstenson et al. (Thorstenson et al., 1998) (also see: http://www-sequence.stanford.edu/protocols/library.html).  The sequence of the linker oligo is 5'pAATTGTTTG3'. The sequences of the oligos used to assemble the adaptor are 5'pTGAGTCACCAAAC3' and 5'pGTGACTCA3'.
  Phage Insert and Control Gene Amplifications:
  	Individual DNA inserts in the phage were amplified using PCR as follows: phage capsids were denatured at 70 degrees C for 5 minutes to release viral DNA.  DNA inserts were amplified using Platinum® PCR Supermix 96 (Invitrogen Life Technologies, Carlsbad, CA) with primers complementary to the vector (primer SK: 5’cgctctagaactagtggatc3’ and primer KS: 5’tcgaggtcgacggtatc3’) following the manufacturer’s instructions.
  	Thirty-four genes from P. brasiliensis were selected for inclusion as known controls on the array.  Genes were chosen based on known or expected expression patterns in M, or Y or during form transition from M to Y.  Template sequences were obtained from GenBank and primer pairs were designed for each gene using Primer 3 software considering an amplicon size of ~1 Kb (Rozen & Skaletsky, 2000).  Nine primer pairs were designed from regulatory gene sequences provided by Dr. Adrian R. Walmsley (Univ. of Durham, UK).
  	All amplified fragments were electrophoresed through 1.0% agarose gels for 12 minutes using an E-Gel® 96 High-Throughput Agarose Electrophoresis System (Invitrogen), and photographed.  Amplicon lengths were estimated using 1 Kb Plus DNA Ladder (Invitrogen).  Amplicons were purified using Nucleofast® 96 PCR Clean-up Kit (BD Biosciences, San Jose, CA).
  Microarray construction:
  	Purified PCR products from the cloned fragments and controls were spotted onto aminosilane coated glass slides (UltraGAPSTM Slides, Corning Inc. Life Sciences, Acton, MA) using custom built robots at the Stanford Microarray Core Facility (www.microarray.org).
  References:
  Oefner, P. J., Hunicke-Smith, S. P., Chiang, L., Dietrich, F., Mulligan, J. & Davis, R. W. (1996). Efficient random subcloning of DNA sheared in a recirculating point-sink flow system. Nucleic Acids Res 24, 3879-3886.
  Thorstenson, Y. R., Hunicke-Smith, S. P., Oefner, P. J. & Davis, R. W. (1998). An automated hydrodynamic process for controlled, unbiased DNA shearing. Genome Res 8, 848-855.
  Rozen, S. & Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132, 365-386.