title: Version 2 DGRC oligonucleotide microarrays
status: Public on Apr 03 2015
contact.city: Kearney
contact.first: Kimberly
contact.middle: Ann
contact.last: Carlson
contact.email: carlsonka1@unk.edu
contact.state: NE
contact.address: 2401 11th Ave, BHS 300
contact.department: Biology
contact.country: USA
contact.institute: University of Nebraska at Kearney
contact.phone: 308-865-1554
organism: Drosophila melanogaster
manufacture_protocol: 1. Indirect labeling with Cy3/Cy5 fluorescent dyes was performed using 12 ug of total RNA per sample using the Superscript Indirect cDNA Labeling System for DNA Microarrays (Invitrogen).
  2. All reagents and buffers used were included in the kit
  3. Following reverse-transcription (RT), the resultant amino-allyl labeled cDNA was incubated with Cy3/Cy5 in DMSO to couple the Cy3 or Cy5 dyes to the cDNA to create fluorescently labeled probes
  4. The CY3 and CY5 labeled probes were purified by gel-exclusion chromatography using SNAP columns (Invitrogen).
  5. Prior to hybridization, the microarray slides were pre-hybridized for 45 minutes at 42°C in 3X SSC solution (3 M NaCl, 0.3 M sodium citrate, 1 mM EDTA) containing 1% bovine serum albumin
  6. The Cy3 and Cy5 probes were mixed together in 40 ul Ambion hybridization buffer #2 (Ambion)
  7. Blocking agents that included poly-dA (20 ug) and Cot-1 DNA (20 ug) were added
  8. Hybridization was performed overnight at 42°C
  9. After hybridization, the slides were washed 2x times with 2.0x SSC, 0.5% SDS at 42 degrees for 15 minutes
  10.  slides were washed then 2x with 0.5x SSC, 0.50% SDS for 15 minutes each