title: SLA/Immune Response/NRSP8 Pig 70 mers Oligonucleotides 3.8K + 13.3K v1
status: Public on Feb 01 2009
contact.city: Jouy-en-Josas
contact.first: Sylvain
contact.last: Marthey
contact.email: abridge@inra.fr
contact.address: Domaine de Vilvert Bat 320
contact.department: Animal Genetics
contact.country: France
contact.institute: INRA
organism: Sus scrofa
description: The oligos set used for this microarray is a mix of the 13,3k generic set from Operon and a 3.8K SLA/Immune response specific set designed by Yu Gao (LREG-INRA) and synthetised by Operon.
manufacture_protocol: 1-1. Oligos from 13K set are arrayed by Operon in Genetix 384-well V-bottom plates. Each well contains 600 pmol of 70-mer oligo.
  1-2. Resuspend oligos in water(20µM).
  1-3. Incubate at room temperature during a night shaking plates to aid resuspension of DNA.
  1-4. Deep freeze and defrost centrifuging plates at 1000 rpm for 16 minutes then 2000 rpm for 3 minutes.
  1-5. Take 5 µl of oligos solution and put it in new Genetix polypropylene v-bottom plates (cat# X7022)
  1-6. Add 5 µL of 1X Corning Printing Buffer to each well and mix.
  2-1. Oligos from 3.8K SLA/Immune response set are arrayed by Operon in Genetix 384-well V-bottom plates. Each well contains 200 pmol of 70-mer oligo.
  2-2. Resuspend oligos in water(20µM).
  2-3. Incubate at room temperature during a night shaking plates to aid resuspension of DNA.
  2-4. Deep freeze and defrost centrifuging plates at 1000 rpm for 16 minutes then 2000 rpm for 3 minutes.
  2-5. Add 5 µL of 1X Corning Printing Buffer to each well and mix.
  3-1. cDNA from Lucidea microarray Scorecard (GE Healthcare) are resuspended in water (100ng/µl).
  3-2. Incubate at room temperature during a night shaking plates to aid resuspension of DNA.
  3-3. Deep freeze and defrost centrifuging tubes at 1000 rpm for 16 minutes then 2000 rpm for 3 minutes.
  3-4. Take 5 µl of solution and put it in new Genetix polypropylene v-bottom plates (cat# X7022). Each cDNA Lucidea scorecard are put 4 times.
  3-5. Add 5 µL of 1X Corning Printing Buffer to each well and mix.
  4. Centrifuge plates at 2000 rpm for 3 minutes.
  5. Oligos are ready for spotting.
  6. Spotting on glass slides (Corning, Ultragaps), with 48 Microspotting pins Stealth 3 on Chipwriter (Virtek), with control of humidity (45-50%).
  7. Fixation of the oligos slides with UV (600 mJoules).
  8. Storage the slides in a vacuum.
  9. After the print run is completed, cover oligos plates with seals and deepfreeze them at -20°C in a protected environment.