Livesey: Agilent arrays

1 Combine 5 μg Cy5- and 5 μg Cy3-labeled samples in a total of 150 μL ddH2O in a 1.5 mL microfuge tube.

2 Add the following in the order indicated to the microfuge tube:

3 Heat samples for 3 minutes at 95°C.

4 Mix contents and quick spin to collect.

5 Immediately transfer the sample tubes to a circulating water bath or heat block at 37°C and incubate for 30 minutes.

6 Spin at 17,900 × g for 1 minute at room temperature to collect the sample.

7 Load a clean gasket slide into the Agilent SureHyb chamber base with the label facing up and aligned with the rectangular section of the chamber base. Ensure that the gasket slide is flush with the chamber base and is not ajar.

8 Slowly dispense 490 μL of hybridization sample onto the gasket well in a "drag and dispense" manner.

9 Place an array “active side” down onto the SureHyb gasket slide, so the numeric barcode side is facing up and the “Agilent” barcode is facing down. Make sure the sandwich-pair is properly aligned. NOTE Use foils and amber tubes to keep samples in the dark as much as possible.

10 Place the SureHyb chamber cover onto the sandwiched slides and slide on the clamp assembly. Hand-tighten the clamp onto the chamber.

11 Vertically rotate the assembled chamber to wet the gasket and assess the mobility of the bubbles. Tap the assembly on a hard surface if necessary to move stationary bubbles.

12 Place assembled slide chamber in rotisserie hybridization oven set to 40°C. Hybridize at 10 rpm for 40 hours. You can use a higher rotation speed (up to 20 rpm) to enhance the overall assay signal intensity.

Table 17 Stock 1x Mix Final Concentration Human Cot-1 DNA (1.0 mg/mL) 50 μL 0.1 mg/mL Agilent Blocking Agent (10x)*

• Supplied in the Agilent Oligo aCGH Hybridization Kit

50 μL 1x Agilent Hybrdization Buffer (2x)* 250 μL 1x