P-CAGE-25088 - P-CAGE-25088

Type
hybridization
Description
1. Hybridization of fragmented cRNA to Arabidopsis2 microarray (G4136A, Agilent)

-Fragmentation of labeled cRNA with in situ hybridization kit plus (5184-3568, Agilent)

-Hybridization Cocktail

1.0 µg / 510 µl Cy3-labeled cRNA (fragmented)

1.0 µg / 510 µl Cy5-labeled cRNA (fragmented)

0.98 x control target (included in the in situ hybridization kit plus, Agilent)

0.49 x fragmentation buffer (included in the in situ hybridization kit plus, Agilent)

0.98 x hybridization buffer (included in the in situ hybridization kit plus, Agilent)

-Hybridization conditions

Hybridization using hybridization chamber (G2534A, Agilent)

60˚C, 16 hr hybridization in a hybridization oven (G2505-80085, Agilent) with rotary shaking (4 rpm)


2. Washing

-Wash Buffer 1:

6x SSC

0.005% Triton X-102 (included in the in situ hybridization kit plus, Agilent)

-Wash Buffer 2:

0.1x SSC

0.005% Triton X-102 (included in the in situ hybridization kit plus, Agilent)

-Washing procedure

Open the hybridization chamber in Wash Buffer1

Wash once in Wash Buffer1 at room temperature for 10 min with moderate stirring

Wash once in Wash Buffer2 at 4˚C for 5 min with moderate stirring

Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
Experiment E-ATMX-8