P-EMBL-143 - P-EMBL-143

Type
hybridization
Description
PREHYBRIDIZATION:

The microarrays were immersed at 42°C in 6xSSC / 0.5%SDS / 1%BSA for 45 min and subsequently washed extensively with ddH2O at room temperature and dried by centrifugation. Prior to hybridization, the spotted PCR products were denatured by immersing the slides at 95°C in ddH2O for 2 min. Excess of liquid was removed from the slides by centrifuging them briefly at 715xg in a microtiter plate centrifuge (Z320, Hermle, Wehingen, Germany).


HYBRIDIZATION:

Prior to hybridization, the purified Cy3- and Cy5-labeled cDNAs were mixed and centrifuged for 10 min at max. speed to precipitate any unsoluble particles. The supernatant was transferred into a fresh tube and combined with 10 µg poly(dA) (Amersham Pharmacia, Uppsala, Sweden) and 4 µg salmon sperm DNA. The mixture was evaporated in a vacuum concentrator 5301 (Eppendorf, Hamburg, Germany) at 60°C. The resulting pellet was dissolved in 40 µl hybridization buffer (50% formamide / 6xSSC / 0.5%SDS / 5x Denhardt's) and denatured by incubation at 95°C for 2 min. The probe was then transferred onto the array under a 24 x 24 mm coverslip and incubated in a humid chamber (GeneMachines, San Carlos, CA, USA) containing 2xSSC drops for providing humidity. Hybridization was performed for 16-18 h in a 42°C waterbath (GFL, Burgwedel, Germany).


WASHING THE HYBRIDIZED SLIDES:

After hybridization the microarrays were washed in a Coplin jar twice in 0.1xSSC / 0.1%SDS for 5 min and twice with 0.1xSSC for 5 min (on an orbital shaker). Slides were briefly immersed in ddH2O. Finally, the washed slides were dried by centrifugation.
Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
Experiment E-EMBL-5