Hybridization FOR CGH

Saturday, May 15, 2004

Description
Hybridization FOR CGH

Procedure
. precipitate DNA and dilute in hybridization solution:

10 l tumor-DNA (Biotin-labeled)

10 l Normal-DNA (Digoxigenin-labeled)

30 l human Cot1-DNA

1 l salmon sperm-DNA

5,1 l 3 M sodiumacetate

150 l 100% ethanol (-20C)

pipette all solutions mentioned above -->store at -80C for 30 min--> centrifuge the tube for 10 min at max. speed at 4C--> remove the supernatent--> wash the pellet with 500 l 70% ethanol --> centrifuge for 10 min at 4C--> remove the supernatent--> dry the pellet in the speed vac centrifuge for 5-10 min--> solve the pellet in 5 l formamide, incubate the probe for 10 min at 37C, add 10 l of master mix

2. Denaturation of the genomic DNA and prehybridization

denature DNA for 5 min at 77C, centrifuge briefly, prehybridiation at 37C for at least 1 h

3. Inspection and denaturation of metaphase chromosomes

during the prehybridization inspect the slides with the metaphase spreads in a phasecontrast microscope, check the quality of the chromosomes and select the best region for hybridization.

add 120 l of 70% FA/ 2xSSCto a cover slip --> place slide with chromosome spreads slowly on the drop of the denaturation solution --> turn the slide --> denature at 77C for 90 sec in a preheated ofen --> place slide vertical to remove cover slip --> place slide in a cuvette with 70% ethanol (ice cold) --> dehydrate by placing the slides in cuvettes with an ascending concentration of ethanol (70, 90 und 100% EtOH, 2 min each) --> air dry the slides by placing the vertically

4. Addition of the hybridization solution to the slide with the metaphases

Centrifuge briefly the tube with the DNA after prehybridisation to remove vapor and fluid from the lid

--> Place slides with the denatured and dry metaphases chromosomes on a heating plate (37C), label slides with case number --> add about 13 l of hybridization solution --> if air bubble are present remove them by the edge of a cover slip --> cover drop with an 18x18 mm cover slip --> seal edges with rubber cement (alternatively place slides in a moist chamber without rubber cement at 37C) --> place slides in a metall box --> place metall box in water bath at 37C --> hybridize for 3 days

Recipes
labeled tumor and normal-DNA (see protocol Nick translation)
salmon sperm DNA, 10 mg/ml (e.g. Promega)
human Cot1 DNA, 1 mg/ml (GibcoBRL, Life Technologies)
3M sodium acetate
100% ethanol, 90% ethanol, 70% ethanol
Formamide (FA), (e.g. from Merck)
Master mix (20% dextransulfate/4xSSC)
SSC (20x)
70% FA/2xSSC for chromosome denaturation (120 l per slide)
18x18 mm2 cover slips, 60x24 mm2 cover slips (e.g. Menzel)
rubber cement (Fixogum, Marabu, D-71732 Tamm)

Supplies

Tips

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Hybridization FOR CGH