P-EMBL-116 - P-EMBL-116
The slides were immersed at 42C in 6x standard saline citrate (SSC) / 0.5% sodium dodecyl sulfate (SDS) / 1% bovine serum albumin (BSA) for 40 minutes and carefully washed in ddH2O at room temperature. The attached PCR-products were then denatured at 95C in ddH2O for two minutes and then air dried. Prior to hybridization, the purified Cy3 / Cy5 labeled cDNA probes were mixed together. 5 µg poly-d-A and 5 µg human Cot1 DNA (both Gibco Invitrogen, Carlsbad, CA) were added and the mixture evaporated in the Vacuum Concentrator 5301 (Eppendorf, Hamburg, Germany) at 45C to complete dryness. The pellet was dissolved in 70 µl hybridization buffer (50% formamide / 6x SSC / 0.5% SDS / 5x Denhardt's) and denatured by incubating at 95C for 2 minutes. The probe was hybridized with the microarray in a humid plastic bag at 42C for 16 hours. After hybridization, slides were washed in 2x SSC / 0.1% SDS for 5 minutes at 42C and then 10 min in 0.2x SSC / 0.1% SDS for 10 minutes at 42C. Slides was then quickly rinsed 3 times in 0.2x SSC / 0.1% SDS. Slides were dried by a brief centrifugation at 715g in a microtiter plate centrifuge (Z320; Hermle, Wehingen, Germany). After a quick dip in isopropanol and a quick spin dry again the slides were scanned immediately.