16 Comparative Genomic Hybridization
207
6. Very slowly add so III of fresh cold Carnoy's fixative (methanol and
acetic acid, 3:I) down the side of the tube. Gently resuspend. Add
ten more SO III volumes, ten 100 III volumes, and ten 200 III volumes
of Carnoy's, pipetting after adding each volume, finally bringing the
total volume to 4 ml. Incubate at 4°C for 20 min.
7. Centrifuge at 1000 rpm for 5 min. Gently pour off supernatant.
8. Repeat the Carnoy's fixing procedure (steps 6-7) twice.
9. After the final centrifugation, resuspend cells in about 200 III of fixative.
10. Arrange the slides on a flat surface and drop 7 III of cell suspension a
slide. Two drops can be placed on one slide - one on each side. Allow
the slides to dry.
11. Examine the slide with a phase contrast microscope to determine if the
cell concentration needs adjustment and assess the success of metaphase production.
12. Age the slides at room temperature for 2 weeks. Then store under nitrogen in sealed plastic bag at -20°e.
I Subprotocol 3
Hybridization
This phase constitutes the heart of the CGH experiment. Test and reference DNAs labeled with different fluorochromes are prepared as a hybridization mixture by precipitating and resuspending them together with
CoT-1 DNA. The metaphases are denatured and incubated together
with the hybridization mixture and then washed. This protocol assumes
DOP-PCR amplified samples as described above.
Materials
- 1x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0.
Reagents
- Master hybridization solution (MM#I): 5 ml formamide, 1 g dextran
sulfate, 1 m120x SSC, water to final volume of7 mt. Suspend the dextran sulfate in formamide and quickly add SSC and water mixing vigorously. Heat 1-2 h at 70°C vortexing intermittently until dissolved. pH
207
6. Very slowly add so III of fresh cold Carnoy's fixative (methanol and
acetic acid, 3:I) down the side of the tube. Gently resuspend. Add
ten more SO III volumes, ten 100 III volumes, and ten 200 III volumes
of Carnoy's, pipetting after adding each volume, finally bringing the
total volume to 4 ml. Incubate at 4°C for 20 min.
7. Centrifuge at 1000 rpm for 5 min. Gently pour off supernatant.
8. Repeat the Carnoy's fixing procedure (steps 6-7) twice.
9. After the final centrifugation, resuspend cells in about 200 III of fixative.
10. Arrange the slides on a flat surface and drop 7 III of cell suspension a
slide. Two drops can be placed on one slide - one on each side. Allow
the slides to dry.
11. Examine the slide with a phase contrast microscope to determine if the
cell concentration needs adjustment and assess the success of metaphase production.
12. Age the slides at room temperature for 2 weeks. Then store under nitrogen in sealed plastic bag at -20°e.
I Subprotocol 3
Hybridization
This phase constitutes the heart of the CGH experiment. Test and reference DNAs labeled with different fluorochromes are prepared as a hybridization mixture by precipitating and resuspending them together with
CoT-1 DNA. The metaphases are denatured and incubated together
with the hybridization mixture and then washed. This protocol assumes
DOP-PCR amplified samples as described above.
Materials
- 1x SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0.
Reagents
- Master hybridization solution (MM#I): 5 ml formamide, 1 g dextran
sulfate, 1 m120x SSC, water to final volume of7 mt. Suspend the dextran sulfate in formamide and quickly add SSC and water mixing vigorously. Heat 1-2 h at 70°C vortexing intermittently until dissolved. pH