P-MEXP-1984 - P-MEXP-1984

Type
hybridization
Description
Hybridisation Materials 1. spotted microarrays on Type 7* mirror slides (Amersham Pharmacia Biotech, RPK2331) 2. Poly-dT35, 1 µg/µL (Metabion) 3. Microarray hybridization buffer (Amersham Pharmacia Biotech, RPK0325) 4. Formamide 5. Glass cover slips, (24×50 to 24×60 mm) 6. 20× SSPE 7. 10% SDS 8. distilled water Protocol Hybridisation Make up the following mixture: 1,17 µg (1,17 µL) Poly-dT35 ¼ Vol. (8,75 µL) Microarray hybridization buffer 50,00% (17,5 µL) Formamide to 35 µL (7,58 µL) water dissolve all of the Cy3 and Cy5-labeled samples in the mixture incubate for 3 min 96 °C chill on ice and leave it there until hybridisation incubate the microarray in 2× SSPE + 0,2% SDS for 30 min, RT dip the microarray a few times in distilled water dry the microarray by soft centrifugation (1100 rpm, 1 min in 50 mL tube) pipet the complete sample onto the microarray cover the microarray carefully with a coverslip; avoid the formation of bubbles! insert the microarray into an incubation chamber incubate over night at 42 °C Washing Preparations: pre-warm a staining chamber with 1× SSC + 0,2% SDS to 55 °C pre-warm two staining chambers with 0,1× SSC + 0,2% SDS to 55 °C pre-warm a staining chamber with 0,1× SSC to 37 °C keep one staining chamber with distilled water at RT Protocol: remove the cover slip from the microarray by carefully dipping in 1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 0,1× SSC + 0,2% SDS wash for 10 min, 55 ° C in 0,1× SSC + 0,2% SDS wash for 1 min, 37 ° C in 0,1× SSC dip five times in distilled water dry by soft centrifugation (1 min, 1100 rpm in 50 mL Falcon tubes)
Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
All experiments using protocol P-MEXP-1984: (E-CAGE-5, E-CAGE-6, E-CAGE-7)