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MORIMASA MATSUTA AND MAYUMI MATSUTA
Materials
- Fluorescent microscope (e.g., Olympus BX type, Nikon) with
suitable single-bandpass or multi-bandpass filter sets (for
FITC, Rhodamin, Texas-Red, Spectrum-Orange, SpectrumGreen, DAPI, PI, etc.).
- Water Baths: 15°C - 74°C
- Vortex
- A bench-top micro centrifuge
- Microcentrifuge tubes: 0.5 ml, 1 ml
- Diamond tipped pen
- Normal male or female metaphase CGH target slides
- Glass cover slips; 18X18 mm, 22X24-60 mm
- Coplin jars
- Micropipettes (variable type:0.5-10111, -200111, 1ml)
- Moist chamber
- Incubator at 37°C
- Hybridization buffer: 50% formamide/l0% dextran sulfate/
2XSSC.
- Purified water
- Formamide: Ultra pure grade (Kodak etc.)
- Rubber cement: Paper bondTM (Kokuyo, Tokyo, Japan)
- Denaturation solution: 50 ml of 70% formamide/2X SSC
- Washing solution: 150 ml of 50% fromamide/2X SSC
- 20X SSC solution
- PN buffer
- Antifade solution, see Recipes in Subprotocol1(FISH), Materials; also commercially available
- DAPI or PI, 100 - 1000 ng in antifade solution
- Ethanol dehydrate solution: concentrations of70, 85 and 100%.
- 3M Sodium acetate
- tumor DNA
- 10xA4mixture:10xA4isO.2mMdATP,dCTP,dGTPin500mM
Tris-HCl (pH 7.8), 50 mM MgCh, 100mM ~-mercaptoethanol,
10011g/ml BSA (nuclease free).
- FITC-12-dUTP and Tex-Red-5-dUTP (DuPont, Boston, MA)
- enzyme mixture containing SNA polymerase I (-2 U) and
DNAse 1(-200 pg) (Gibco BRL, Gaithersburg, MO)
- DNA polymerase I (Promega, Madison, WI)
- proteinase K solution (O.ll1g/ml in 20 mM Tris-HC1!2mM
CaCh, pH 7.5)
MORIMASA MATSUTA AND MAYUMI MATSUTA
Materials
- Fluorescent microscope (e.g., Olympus BX type, Nikon) with
suitable single-bandpass or multi-bandpass filter sets (for
FITC, Rhodamin, Texas-Red, Spectrum-Orange, SpectrumGreen, DAPI, PI, etc.).
- Water Baths: 15°C - 74°C
- Vortex
- A bench-top micro centrifuge
- Microcentrifuge tubes: 0.5 ml, 1 ml
- Diamond tipped pen
- Normal male or female metaphase CGH target slides
- Glass cover slips; 18X18 mm, 22X24-60 mm
- Coplin jars
- Micropipettes (variable type:0.5-10111, -200111, 1ml)
- Moist chamber
- Incubator at 37°C
- Hybridization buffer: 50% formamide/l0% dextran sulfate/
2XSSC.
- Purified water
- Formamide: Ultra pure grade (Kodak etc.)
- Rubber cement: Paper bondTM (Kokuyo, Tokyo, Japan)
- Denaturation solution: 50 ml of 70% formamide/2X SSC
- Washing solution: 150 ml of 50% fromamide/2X SSC
- 20X SSC solution
- PN buffer
- Antifade solution, see Recipes in Subprotocol1(FISH), Materials; also commercially available
- DAPI or PI, 100 - 1000 ng in antifade solution
- Ethanol dehydrate solution: concentrations of70, 85 and 100%.
- 3M Sodium acetate
- tumor DNA
- 10xA4mixture:10xA4isO.2mMdATP,dCTP,dGTPin500mM
Tris-HCl (pH 7.8), 50 mM MgCh, 100mM ~-mercaptoethanol,
10011g/ml BSA (nuclease free).
- FITC-12-dUTP and Tex-Red-5-dUTP (DuPont, Boston, MA)
- enzyme mixture containing SNA polymerase I (-2 U) and
DNAse 1(-200 pg) (Gibco BRL, Gaithersburg, MO)
- DNA polymerase I (Promega, Madison, WI)
- proteinase K solution (O.ll1g/ml in 20 mM Tris-HC1!2mM
CaCh, pH 7.5)