title: population genetic approach to host patheogen response
status: Public on May 26 2006
contact.city: Albuquerque
contact.first: John
contact.middle: Joseph
contact.last: Rushton
contact.email: rushton@unm.edu
contact.state: NM
contact.address: MSC10 5550, 1 University of New Mexico
contact.department: Internal Medicine
contact.country: USA
contact.institute: University of New Mexico, School of Medicine
contact.phone: 505-272-0738
organism: Homo sapiens
manufacture_protocol: Oligonucleotide printing, microarray slide preparation and processing :Oligonucleotide libraries are printed on poly L Lysine coated microscope slides which are blocked prior to hybridization. Prior to printing, the microscope slides (Gold Seal, VWR, So. Plainfield, NJ) are cleaned with 2.5 M NaOH in 61% ethanol, washed thoroughly, and then coated with 0.01% poly l lysine (Sigma, St. Louis, MO) in filtered 10% phosphate buffered saline without calcium or magnesium (Hyclone, Logan, UT). After coating, excess poly l lysine is rinsed off and the slides are vacuum dried and stored at least 2 weeks prior to printing. The mouse ver 1.1 library of 70mer oligonucleotides (Operon, Alameda,  CA) is resuspended at 40uM in 3xSSC in 384 well v bottom plates (Genetix, Beaverton, OR) prior to printing 100 slides on an OmniGrid Microarray printer (GeneMachines, San Carlos, CA). In brief, the printing conditions are 60% humidity, 16 Majer pins (Majer Precision Engineering, Tempe, AZ), 24 column by 22 row subarrays,  8448 features per array including controls, duplicate arrays per slide, 0 millisecond print time, and 22  384 well plates per print run.  Between each sample, pins are cleaned with two loops of 3 second sonication, 2 second washing and 4 second drying per loop.  Printed controls include 70mer oligonucleotides specific for constitutively expressed housekeeping genes (e.g. actin, 28s rRNA), 3xSSC printing buffer, blanks, murine genomic DNA, and B. subtilis gene pcr products (American Type Culture Collection, Rockville MD).  After printing, the microarray slides are rehydrated, snap dried at 105oC,  UV crosslinked at 65 mjoules (Stratalinker, Stratagene, LaJolla, CA) and blocked with 43mM sodium borate (Fisher, Fair Lawn, NJ) and 157mM succinic anhydride (Aldrich, Milwaukee, WI) in 1-methyl 2-pyrolidinone (Aldrich, Milwaukee, WI).