TITLE: aCGH Hybridization & wash
AUTHORS: Hendrik F van Essen

[STEPS]
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Turn on hybridization oven Turn on heat block Turn on heat block
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Mix Cy3 and Cy5 samples
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Mix the following components in 1.5 centrifuge tubes*     Cy3/Cy5 gDNA mixture		  39 µl*     Cot-1 DNA (1.3 µg/µl)		            5 µl*     Agilent 10x blocking agent	  	  11 µl*     Agilent 2x Hybridization buffer	  55 µl                        Total				110 µl
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Mix sample by pipetting, by careful not to create any bubbles.
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Spin down with maximum speed for a few seconds.
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Heat for  at and incubate immediately thereafter for  at .
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Leave samples in heat block, spin down for  at maximum speed.
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Load gasket slide in hybridization chamber and dispense 100 µl hyb mix in a drag and drop manner (use a 200 µl pipette, be careful not to create any bubbles)
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Place microarray slide – active side down – and assemble chamber.
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Check for immobile air bubbles by rotating the assembled hybridization chamber. If necessary, slam side of the chamber on table to loosen the air bubbles.
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Place assembled chamber in the hybridization oven ( at 20 rpm) and make sure to balance with empty chambers.
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Hybridize for , at 20 rpm.
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Turn on Agilent Slide Scanner
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Wash buffer dishes with distilled water and blow dry with pressurized air.
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Add buffers to dishes and have tin foil covering ready.-	Wash a maximum of 5 Agilent slides at the same time.Dish	Method	                Buffer		Temp (ºC)	Time (min)#1	        Disassembly    	Buffer 1	        RT		            -#2	        1st wash step	Buffer 1	        RT		            5	(stirrer)#3	        2nd wash step	Buffer 2	        37		            1	(stirrer & hot plate)#4	        Dry step	                Acetonitrile	RT		            1
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Separate gasket slide from microarray slide in dish #1 (buffer 1)
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Place slides in holding rack and was in dish #2 for , rotation of stirrer is switched ON!
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Cover dish with tin foil.
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Start the "Scan Control Software"For Agilent 180k, select the “AgilentG3_CGH” Option
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Click on “Set Values” to confirm all settings
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Move holding rack to dish #3 (buffer 2) and wash for , rotation of stirrer and heating is switched ON!
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Move holding rack to dish #4 (acetonitrile) leave for .
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Remove slide gently with tweezers, thus preventing the formation of droplets.
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Place slide in holder and load into the carousel of the scannerAgilent Slide Scanner scans through the back of the slide (this side must contain the numeric barcode)
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Open “feature extraction” and check TIFF file
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Click Scan Slot m-n on the Scan Control main windowThe letter m represents the Start slot where your first slide is located, and the letter n represents the End slot where your last slide is located.
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Start the scan