title: PGT- 30K Human Array v1
status: Public on Mar 24 2010
contact.city: Nottingham
contact.first: Patrick
contact.middle: Jason
contact.last: Tighe
contact.state: Nottinghamshire
contact.address: University Hospital
contact.department: School of Molecular Medical Sciences
contact.country: United Kingdom
contact.institute: The University of Nottingham
organism: Homo sapiens
description: spotted oligonucleotides from MWG 30K sets A, B and C oligo sets
manufacture_protocol: The Oligoset (in Genetix 7020 polypropylene plates) was printed at 20µM concentration, in 3xSSC, 1.5M Betaine, using a Genomic solutions Microgrid II 600 Arrayer and  microspot 2500 split pins at 65% humidity. Prints were made onto Schott Nexterion A+ barcoded slides (APS coated) using 48 pins in a 4x12 array to give 48 subgrids, each of 26 columns x26 rows of possible features.  Target spot diameter was 80 micrometers.   The GE-Lifesciences Universal scorecard Oligo set were printed onto each subgrid for  QC and hybridization controls. Cy3- random 9mer-NH oligonucleotideLanding lights were printed at corners of each subgrid
  Slide post-processing: Slides were then rehydrated whilst suspended upside-down in a humid chamber (80% relative humidity) for 10 minutes, followed by snap drying for 10 seconds at 120°C on a hot plate.  Slides were then baked for 3h at 80°C and stored at room temperature under dessication and vaccum untill required.  Prehybridization, each slide was washed sand blocked to remove excess oligo and block the surface by immersion in blocking solution (5xSSC 0.2%SDS 1%BSA using warm Ultra Pure Water),  Filter the blocking solutionbefore use.Block for 10 minutes with vigorous shaking or stirring of the solution..  Prepare three more wash stations, two containing Ultra Pure Water and one containing 100% Ethanol, seal the containers to prevent evaporation.  Prepare a  plate rotor on a benchtop centrifuge and set to 600rpm for two minutes.  Prepare a slide rack containing blank slides as a balance.  After the 10 minutes dunk the slides into the first wash station of Ultra Pure Water three times, the second twice then into the 100% Ethanol.  Carry this container and slides over to the centrifuge while still in the ethanol and coentrifuge to  dry. The slides should be preferably hybridized on the day of blocking, as they are not suitable for storage for more than a couple of days once blocked.