title: DUMRU Mouse 12K v1.0
status: Public on Sep 09 2010
contact.city: Waurn Ponds
contact.first: Sean
contact.last: McGee
contact.state: Victoria
contact.address: 75 Pigdons Rd
contact.department: Metolic Research Unit, School of Medicine
contact.country: Australia
contact.institute: Deakin University
organism: Mus musculus
description: ONLY CLONES OF INTEREST HAVE BEEN SEQUENCED AND IDENTIFIED
manufacture_protocol: 1. Individual cDNA clones were randomly selected from a mouse skeletal muscle cDNA library (5?-Stretch Plus, Clontech, Palo Alto, CA, USA) using a BioPick automated colony picker (BioRobotics Woburn, MA, USA)
  2. The cDNA inserts were PCR amplified using primers and conditions recommended by the library manufacturer (Clonetch) and the PCR products were visualized by agarose gel electrophoresis but not sequenced and hence each clone identity remained unknown
  3. Amplified cDNAs were then purified using an ArrayIT PCR Purification Kit and resuspended in 30µl of ArrayIt spotting solution (TeleChem International, Sunnyvale, USA)
  4. 5µl of resuspended cDNA was transferred to 384 well Uniplates (Whatman Inc, Clifton, USA)
  5. cDNA was printed onto 40 microarray slides using an SDDC2 robotic arrayer (Engineering Services Inc, Toronto, Canada)
  6. The arrayer was set up to print 32 cDNA spots per cycle with ArrayIt Stealth SMP3 quill tipped microarray pins (TeleChem, International Inc, Sunnyvale, USA)
  7. Spacing between cDNA spots was 200 microns
  8. Each pin drew 0.25µl of cDNA, tapped a blotting slide twice and deposited approximately 0.6nl on each SuperAmine microarray glass slide (TeleChem International, Sunnyvale, USA)
  9. Pins were washed in distilled water and air-dried automatically between cycles
  10. Humidity was kept between 55 and 65% during printing
  11. Slides were stored at 35% humidity for 36 hours to dry before fixing
  12. cDNA was fixed to the slides using the following procedures.
  13. Slides were placed in an oven at 80oC for 1 hour, and then allowed to cool to room temperature
  14. Slides were placed DNA side down in a humidity chamber (Sigma, St Louis, USA) for 60 seconds to rehydrate the DNA before snap drying them on a heating block set to >100oC
  15. cDNA was fixed to the glass slide by UV cross-linking at 60mJ
  16. Slides were washed with constant agitation in the following separate solutions: 0.2% SDS, water, water, boiling water, and again in 0.2% SDS, water, water for 2 minutes
  17. Immediately after the last wash, slides were dried by centrifugation at 800xg for 5 minutes
  18. Slides were then stored until ready for hybridisation experiments