P-TABM-316 - P-TABM-316

Type
hybridization
Description
CATMA reflective slides (CATMAv2.3 or CATMAv2.4, 23K, printed by VIB) were humidified by holding the array over near boiling water for 1-2 min and then letting them dry flat. The slides were x-linked at 50 mJ and then immediately pretreated for 30-60 min with 2x SSPE/0.2%SDS at room temperature, protected from light. The slides were rinsed by being dipped a few times in distilled water at RT and immediately dried by centrifugation. The slide was placed in a TeleChem manual hybridization cassette with a 25x60 LifterSlip coverslip and 15 ul of 3x SSC for each groove. The probe was diluted to a final volume of 90uL as follows: cDNA (combination of 40 pmol ± 5 pmol of Cy5-labeled cDNA and 40 pmol± 5 pmol of Cy3-labeled cDNA); yeast tRNA (0.2ug/ul final volume); 3% SSC; 0.38% SDS; 50% formamide. The probe mixture was boiled for 1 min, and centrifuged for 1 min. The probe was added to the microarray chip as quickly as possible, using capillary action to cover the area under the 25x60 lifterslip. The microarray cassette was sealed and submerged in a covered 42oC water bath for approximately 16 h. The slide was washed for 10 min in 1xSSC, 0.2% SDS (55oC) (coverslip was removed in first wash solution); 10 min in 0.1x SSC, 0.2% SDS (55oC); 10 min in 0.1x SSC, 0.2% SDS (55oC); 1 min in 0.1x SSC (37oC); and 1 min in 0.1x SSC (25oC) then dried immediately by centrifugation (2 min at 2600 rpm). It was stored in a dark container and scanned as soon as possible.
Links
Experiment E-CAGE-189