P-EMBL-174 - P-EMBL-174

Type
hybridization
Description
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. 10 ug poly-d-A and 10 ug human Cot1 DNA (both Gibco Invitrogen, Carlsbad, CA) were added and the mixture evaporated in the Vacuum Concentrator 5301 (Eppendorf, Hamburg, Germany) at 60C to complete dryness. The pellet was dissolved in 50 ul 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 chamber in a water bath at 42C for 16 hours. After hybridization, slides were washed in 0.1x SSC / 0.1% SDS for 10 minutes and then twice with 0.1x SSC for 5 minutes at 37C with 130 rpm on an orbital shaker (Gio Gyrotory Shaker, New Brunswick Scientific, Edison, N.J.). Slides were dried by a brief centrifugation at 715 g in a microtiter plate centrifuge (Z320; Hermle, Wehingen, Germany) and scanned immediately.
Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
Experiment E-EMBL-4