title: NSWCAG 16K
status: Public on Apr 23 2009
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
manufacture_protocol: The cDNA clones were grown overnight 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 lg/ml ampicillin, in 96-well microtiter plates. These plates were replicated in 150 µl of fresh medium and grown overnight. The replicate plate was stored at 80 oC after the addition of 100 µl 80% (v/v) glycerol. Plasmid preparations were made from cultures following standard alkaline lysis procedures, and the inserts were amplified by PCR using the M13/pUC forward and reverse sequencing primers (New England Biolabs, Beverly, Mass.). For quantitative and qualitative evaluation of the amplification procedure, 1 µl 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 µl 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 CMTGAPS-coated microscope slides (Corning, USA) using an Omnigrid Microarrayer (Genemachines, San Carlos, Calif.) with ChipMaker2 quill pins (TeleChem, Sunnyvale, Calif.). Prior to hybridisation the slides were baked at 80 oC for 2 h.