title: CSIRO NSW-AGC Wheat Barley 22K Array
status: Public on Jul 25 2006
contact.city: Canberra
contact.first: Andrew
contact.last: Spriggs
contact.email: andrew.spriggs@csiro.au
contact.state: ACT
contact.address:  GPO Box 1600
contact.department: Bioinformatics Group
contact.country: Australia
contact.institute: CSIRO Plant Industry
contact.phone: 612-6246-5193
organism: Hordeum vulgare
  Triticum aestivum
description: 22K Wheat cDNA Array
manufacture_protocol: Clones were selected from the following cDNA libraries for the construction of the microarray. These libraries were a non-normalised endosperm library from T. aestivum cv Wyuna (Clarke et al. 2000) and two normalised endosperm libraries; one from T. aestivum cv Hartog and the other from Hordeum vulgare cv Himalaya.  Approximately 6,000 EST clones, mapped to the wheat genome, kindly supplied by O. Anderson, Western Region Research Centre, USDA, 800 Buchanan St., Albany, CA 9471 USA. A unigene set of 9,000 EST clones from Bristol University (see cerealsdb.uk.net website for details of the libraries used). Kindly supplied by Keith J. Edwards, School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK. A collection of 400 ESTs from drought stressed wheat seedlings, kindly supplied by Heather Way and Xue Gang-Ping, CSIRO PI, St. Lucia, Brisbane, Australia. The non-redundant set of cDNA clones were grown over night in 250 ml TYPGN medium, (20 g tryptone, 19 g yeast extract, 10 ml 80% (v/v) glycerol, 5 g Na2HPO4, 10 g KNO3 per litre) supplemented with 100 ?g/ml Ampicillin, in 96-well microtiter plates. These plates were replicated in 150 ?l of fresh medium and grown over night. The replicate plate was stored at -80oC after the addition of 100 ?l of 80% (v/v) glycerol. Plasmid preparations were made of the primary plate, following standard alkaline lysis procedures, and the inserts were amplified by PCR using the M13/pUC forward and reverse sequencing primers (New England Biolabs, Inc.). For quantitative and qualitative evaluation of the amplification procedure, 1 ?l of the PCR reaction was run on a 2% (w/v) agarose gel in Tris-borate buffer pH 8 and the image recorded. The remainder of the PCR products were ethanol-precipitated, resuspended in 6 ?l of 50% (w/v) dimethyl sulfoxide and transferred from the 96-well plates to 384-well microtiter plates for printing. The PCR products were printed onto CMT-GAPSTM coated microscope slides (Corning USA) using an Omnigrid Microarrayer (Genemachines, San Carlos, CA) with ChipMaker2 quill pins (TeleChem International Inc., Sunnyvale, CA). Prior to hybridisation the slides were baked at 80 OC for 2 hours.