P-EMBL-38 - P-EMBL-38

Type
hybridization
Description
The slides were immersed at 42°C 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 95°C in ddH2O for two minutes and then air dried. Prior to hybridization, the purified Cy3 / Cy5 labeled cDNA probes were mixed together. 20 µg poly-d-A and 20 µg human Cot1 DNA (both Gibco Invitrogen, Carlsbad, CA) were added and the mixture evaporated in the Vacuum Concentrator 5301 (Eppendorf, Hamburg, Germany) at 60°C to complete dryness. The pellet was dissolved in 50 µl hybridization buffer (50% formamide / 6x SSC / 0.5% SDS / 5x Denhardt's) and denatured by incubating at 95°C for 2 minutes. The probe was hybridized with the microarray in a humid chamber in a water bath at 42°C 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 37°C with 130 RPM on an orbital shaker (Gio Gyrotory Shaker, New Brunswick Scientific, Edison, N.J.). Slides were dried by a brief centrifugation at 715g in a microtiter plate centrifuge (Z320; Hermle, Wehingen, Germany) and scanned immediately.
Parameters
chamber type, quantity of label target used, temperature, time, volume
Links
All experiments using protocol P-EMBL-38: (E-EMBL-1, E-EMBL-3)