The following information is also available in  Automatic Hybridization.

This section describes the hybridization of ULS labeled genomic DNA on CodeLink DNA arrays using a Tecan HS 4800 hybridization station. Other slides or hybridization stations may be used as well; however, volumes and buffers have been optimized by Simon Joose et al. (BMC Cancer. 2007; 7: 43) at the Netherlands Cancer Institute for the Tecan system specifically, using a 20 mm by 63.5 mm module.

MATERIALS:

Hybridization buffer (filtered with a 0.45 µm filter): 50% formamide 15% dextran sulphate (USB, 14489), Mol weight of 50,000 Da 0.1% Tween 20 2x SSC 10 mM Tris pH 7.4 25 mM EDTA 3 M sodium acetate pH 5.2 Human C0t-1 DNA 1 mg/ml 10 mg/ml Herring sperm DNA 100% and 70% ethanol Yeast tRNA, dissolve to 100 ìg/ìl in H₂O.

PROTOCOL

A. Precipitation

• Pipette into micro centrifuge tubes: 

Mix each tube gently and precipitate at -20°C for 30 minutes to overnight.

B. Preparation of the probe

• Pre-heat hybridization buffer at 37°C
• Spin the precipitated DNA at 4°C for 30 min at 14000 rpm
• Remove supernatant, add 500 ìl 70% EtOH, and re-spin at 14000 rpm for 5 min
• Remove supernatant with a small tip. Make sure no EtOH is left in the tube
• Resuspend the tubes as follows:

Tube 1 (sample):

• 140 µl hybridization buffer
• 10 µl yeast tRNA

Tube 2 (pre-hybridization buffer):

• 150 µl hybridization buffer

This is best done by adding the hybridization buffer and leaving the tube for at least one hour mixing at 650 rpm a 42°C in a Thermomix heat block or for 2-3 hours in a 42°C heat block while vortexing the probe every now and then.
Make sure everything is resuspended properly before you carry on with the experiment.

• Denature the tubes for 10 minutes at 95°C
• Pulse spin the tube

C. Hybridization and washing

Before denaturing the probes, make sure all the washing solutions are fresh and available for the hyb-station at the right channels.

Wash buffers:

• 2x SSC, pH 7
• 2x SSC, 0.1% SDS
•  0.1x SSC

While denaturing the probe, put the slides to be hybridised onto the machine with the labels towards you and close the block. Start the program for aCGH hybridization. Start injection of the probes when the machine asks for it.

While pre-hybridization is running put sample at 37°C to block repetitive sequences.

Hybridization program:

Step 1: Wash at 37°C, 2x SSC (pH 7), 1 wash run for 30 sec, no soak 
Step 2: Probe injection (pre-hyb mix) at 37°C 
Step 3: Hybridization at 37°C for 01:00:00, Agitation Frequency: high
Step 4: Wash at 37°C, 2x SSC (pH 7), 1 run, wash time 15 sec, no soak
Step 5: Probe injection (sample) at 37°C
Step 6: Hybridization at 37°C for 69:00:00, Agitation Frequency: high
Step 7: Wash at 37°C, 2x SSC, 0.1% SDS, 12 runs, wash time 1 min, soak time 1 min
Step 8: Wash at 68°C, 2x SSC, 0.1% SDS, 6 runs, wash time 1 min, soak time 1 min
Step 9: Wash at 68°C, 2x SSC (pH 7), 2 runs, wash time 1:30 min, soak time 1 min
Step 10: Wash at 23°C, 0.1x SSC, 1 wash run for 1:30 sec, no soak
Step 11: Slide drying step at 30°C for 2 min, final manifold cleaning using N2 gas

Collect slides from the machine when the program is finished.

The following information is also available in  Automatic Hybridization.

This section describes the hybridization of ULS labeled genomic DNA on CodeLink DNA arrays using a Tecan HS 4800 hybridization station. Other slides or hybridization stations may be used as well; however, volumes and buffers have been optimized by Simon Joose et al. (BMC Cancer. 2007; 7: 43) at the Netherlands Cancer Institute for the Tecan system specifically, using a 20 mm by 63.5 mm module.

MATERIALS:

Hybridization buffer (filtered with a 0.45 µm filter): 50% formamide 15% dextran sulphate (USB, 14489), Mol weight of 50,000 Da 0.1% Tween 20 2x SSC 10 mM Tris pH 7.4 25 mM EDTA 3 M sodium acetate pH 5.2 Human C0t-1 DNA 1 mg/ml 10 mg/ml Herring sperm DNA 100% and 70% ethanol Yeast tRNA, dissolve to 100 ìg/ìl in H₂O.

PROTOCOL

A. Precipitation

• Pipette into micro centrifuge tubes: 

Mix each tube gently and precipitate at -20°C for 30 minutes to overnight.

B. Preparation of the probe

• Pre-heat hybridization buffer at 37°C
• Spin the precipitated DNA at 4°C for 30 min at 14000 rpm
• Remove supernatant, add 500 ìl 70% EtOH, and re-spin at 14000 rpm for 5 min
• Remove supernatant with a small tip. Make sure no EtOH is left in the tube
• Resuspend the tubes as follows:

Tube 1 (sample):

• 140 µl hybridization buffer
• 10 µl yeast tRNA

Tube 2 (pre-hybridization buffer):

• 150 µl hybridization buffer

This is best done by adding the hybridization buffer and leaving the tube for at least one hour mixing at 650 rpm a 42°C in a Thermomix heat block or for 2-3 hours in a 42°C heat block while vortexing the probe every now and then.
Make sure everything is resuspended properly before you carry on with the experiment.

• Denature the tubes for 10 minutes at 95°C
• Pulse spin the tube

C. Hybridization and washing

Before denaturing the probes, make sure all the washing solutions are fresh and available for the hyb-station at the right channels.

Wash buffers:

• 2x SSC, pH 7
• 2x SSC, 0.1% SDS
•  0.1x SSC

While denaturing the probe, put the slides to be hybridised onto the machine with the labels towards you and close the block. Start the program for aCGH hybridization. Start injection of the probes when the machine asks for it.

While pre-hybridization is running put sample at 37°C to block repetitive sequences.

Hybridization program:

Step 1: Wash at 37°C, 2x SSC (pH 7), 1 wash run for 30 sec, no soak 
Step 2: Probe injection (pre-hyb mix) at 37°C 
Step 3: Hybridization at 37°C for 01:00:00, Agitation Frequency: high
Step 4: Wash at 37°C, 2x SSC (pH 7), 1 run, wash time 15 sec, no soak
Step 5: Probe injection (sample) at 37°C
Step 6: Hybridization at 37°C for 69:00:00, Agitation Frequency: high
Step 7: Wash at 37°C, 2x SSC, 0.1% SDS, 12 runs, wash time 1 min, soak time 1 min
Step 8: Wash at 68°C, 2x SSC, 0.1% SDS, 6 runs, wash time 1 min, soak time 1 min
Step 9: Wash at 68°C, 2x SSC (pH 7), 2 runs, wash time 1:30 min, soak time 1 min
Step 10: Wash at 23°C, 0.1x SSC, 1 wash run for 1:30 sec, no soak
Step 11: Slide drying step at 30°C for 2 min, final manifold cleaning using N2 gas

Collect slides from the machine when the program is finished.