Array CGH Labeling and Hybridization Protocol Molecular Cytogenetics Group Department for Human Molecular Genetics Max Planck Institute for Molecular Genetics, Berlin Last update: 12/13/2005 For questions or comments please contact ullmann@molgen.mpg.de Equipment: Thermocycler with heated lid Waterbath Slide Booster (Advalytix; distributed by Implen, Germany) DNA arrays (this protocol is calculated for a hybridization area of 24x60mm) Coverslips (we do not use lifterslips) Invitrogen: Bioprime Array CGH Kit   CatNo 18095-011 (purification module included) tRNA  CatNo 15401-011 Herring sperm DNA (10mg/ml) CatNo  15634-017 Roche: Cot1-DNA CatNo  1581074 Merck: Sodium acetate CatNo 1.06268.1000 SDS (10%) CatNo 1.06022.1000 Formamide CatNo 1.09684.1000 USB: Dextran sulfate CatNo US70796 Amersham/GE Healthcare: Cy3 dUTP CatNo PA53022 Cy5 dUTP CatNo PA55022 Sigma: BSA CatNo  A-2153 Buffers: FDST, pH7: 0,8g dextran sulfate in 5ml formamide, add 1ml 20xSSC and H2O to 7ml. Heat to 70°C and mix vigorously to dissolve Wash solution1: 50% formamide, 2xSSC, 0,1%SDS PN: 100mM sodium phosphate (pH 8,0) and 0,05% NP40 Pre-hybridization: 25% formamide, 4xSSC and 0,1%SDS We use millipore water for the preparation of  wash and pre-hybridization buffers Pre-hybridization of slides + add 0,3g BSA and 200µl herring sperm DNA to 60ml pre-hybridization buffer and prewarm to 42°C + incubate slides in this supplemented pre-hybridization buffer for 1h at 42°C and rinse with water afterwards + dry slides by centrifugation for 5min with about 150g at room temperature  pre-hybridization should be done shortly before hybridization Labeling Test DNA Reference DNA + add 1µg of DNA in a total volume of 21µl a.d. to each of two tubes +  add 1µg of DNA in a total volume of 21µl a.d. to each of two tubes xxxxxxccc ccccccccc • • + add 20µl of 2,5x Random Primer Solution (Kit) to each tube + mix and spin down + denature DNA by heating it up to 95°C for 5min + put immediately on ice for 5min + spin down + add the following to each tube: cc10x dUTP Nucleotide Mix (Kit)........5µl ccCy3-dUTP or Cy5-dUTP.................3µl ccExo-Klenow Fragment (Kit)..............1µl + mix and spin down + incubate for two hours at 37°C + to each tube add 5µl Stop Solution (Kit) and mix well + purify samples with the Purification Module according to the manufacturers recommendations + elute with 50µl a.d. + pool the labeled probes (final volume 200µl of labeled DNA) bbbbbbbbbbbbbbbbbbbb Hybridization + add the following to the pooled DNA (200µl) cc500µg of Cot1-DNA in a (reduced) volume of 100µl cc30µl sodium acetate 3M, pH5,5 cc825µl EtOH + precipitate at -20°C for at least 2h or overnight + centrifuge at 4°C for 30min + discard supernatant + wash pellet with 100µl 70% EtOH + centrifuge at 4°C for 15min + discard supernatant and let pellet dry + dissolve pellet in 4µl tRNA (100µg/µl) and 8µl 10%SDS + mix well + add 28µl FDST (final concentration of dextran sulfate is 8%) + mix well + denature DNA by heating it up to 70°C for 15min + incubate for 2h at 42°C for preannealing + hybridize under a coverslip for 20-24h at 42°C using a Slide Booster (3:7 mixing/pausing) Washing + prewarm wash solution 1 to 42°C + immerse slides into 2xSSC and let the coverslips fall off (or carefully remove them) + wash slides for 15min in wash solution 1 at 42°C + shortly immerse in PN buffer (room temperature) + immerse slides in a second coplin jar with fresh PN buffer and put it on a rocking table for 10min at room temperature + wash slides in PBS for 30sec at room temperature + immerse slides for a few seconds in a.d. and dry by centrifugation for 5min with about 150g at room temperature + scan slides immediately Volumes of the wash buffers are dependent on the coplin jar used The protocol is provided "as is". No warranty.