CGH Protocols （三）

Hybridization

reagents: 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 µl per slide) 18x18 mm2 cover slips, 60x24 mm2 cover slips (e.g. Menzel) rubber cement (Fixogum, Marabu, D-71732 Tamm)

Steps:

1. precipitate DNA and dilute in hybridization solution:

10 µl tumor-DNA (Biotin-labeled)

10 µl Normal-DNA (Digoxigenin-labeled)

30 µl human Cot1-DNA

1 µl salmon sperm-DNA

5,1 µl 3 M sodiumacetate

150 µl 100% ethanol (-20°C)

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

2. Denaturation of the genomic DNA and prehybridization

denature DNA for 5 min at 77°C, centrifuge briefly, prehybridiation at 37°C 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 µl 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 77°C 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 (37°C), label slides with case number --> add about 13 µl 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 37°C) --> place slides in a metall box --> place metall box in water bath at 37°C --> hybridize for 3 days

DNA detection

Reagents:

Formamide (FA) / 2x SSC (1:1)

0.1x SSC

4x SSC / 0.1% Tween20

3% BSA in 4x SSC / 0.1% Tween 20

DAPI (4.6-diamidino-2phenylindole dihydrochloride): stock solution 0.2 mg/ml, dissolve 1:5000 in aqua bidest, keep solution in use in a light protected cuvette

anti-Dig-Rhodamin, 200 µg/ml (Boehringer Mannheim)

Fluorescein-Avidin-dcs, 1 mg/ml (Vector Laboratories, catalogue-n° A-2011)

Fluorochrome solution:10 µl anti-Dig-Rhodamin + 5 µl Fluorescein-Avidin in 1000 µl 3% BSA/4xSSC/0.1%Tween

DABCO (1,4-diazabicyclo2.2.2octane): 2.3% w/v in Glycerol/Tris, e.g. dissolve 23 mg DABCO in 10 ml Glycerol/Tris pH (9 vol Glycerol plus 1 vol Tris 0.2M, pH8)

24x60 mm2 cover slips (e.g. Menzel)

Steps:

Wash slides 3 x 3 min at 37°C in FA / 2x SSC (new cuvette). Wash slides 3 x 2 min at 60°C in 0.1x SSC (new cuvette). Keep slides in 4x SSC / 0.1% Tween20 at 37°C (new cuvette) until the next step. Cover each slide with 125 µl 3% BSA solution and a 24x60 mm2 cover slip. Incubate 15 min in a moist chamber at 37°C (= blocking step). Remove cover glasses and place slides in 4x SSC / 0.1% Tween20 (new cuvette) up to the next step. Centrifuge fluorochromes 3 min at 14000 rpm at 4°C Prepare fluorochrome solution (see above) Cover each slide with 125 µl of the fluorochrome solution and a 24x60 mm2 cover slip. Incubate 20 min in a moist chamber at 37°C. Remove cover slips and wash slides 3 x 3 min at 45°C in 4x SSC / 0.1% Tween20. Incubate slides 5 min in DAPI solution (room temperature). Mount the slides using DABCO (use about 35 µl DABCO per slide). Store slides at 4°C under dark conditions until microscopy.