31 Multicolor FISH Techniques for the Detection of Inter- and Intrachromosomal Rearrangements
413
- Hybridization Master Mix 2.1: 14.3% w/v dextran sulfate, 78.6% formamide, 2.9xSSC, pH 7.0. Add 1.45 mI20xSSC, 0.7 ml sterile distilled
water and 1.43 g dextran sulfate to 7.86 ml formamide. Heat to 70°C to
dissolve dextran sulfate. Aliquot in 1.5 ml tubes and store at -20°C.
- Mouse anti-FITC (DAKO), 430 Ilg/ml in sterile distilled water, dilute
1:200 in PNM buffer.
- 1x PBS/50 mM MgClz: add 50 mIlO x PBS and 25 ml 1 M MgClz to 425
ml sterile distilled water.
- IOxPBS (Dulbecco's PBS): dissolve 95.5 g PBS Dulbecco in 1000 ml
distilled water and sterilize the solution. Store at room temperature.
- Pepsin solution: prepare pepsin stock solution of 10 mg/ml in sterile
distilled water. Add 10-50 III stock solution to 100 ml 0.01 M HC!. Prewarm at 37°C in water bath, prepare fresh daily.
- PN buffer: (Na2HP04-7 hydrate/NaH 2 P0 4 -1 hydrate, pH 8, 0.05% nonidet P-40)
- PNM buffer: 5% nonfat dry milk, 0.1% NaN 3 in PN buffer
- Proteinase K solution: prepare proteinase K stock solution of 10 mg/ml
in sterile distilled water. Add 3 III stock solution to a buffer containing
2 mIl M Tris/HCI pH 7.5,0.2 mIl M CaClz and 97.8 ml distilled water.
Prewarm at 37°C in water bath, prepare fresh daily.
- Rhodamine-labeled sheep anti-digoxigenin (Roche Molecular Biochemicals), 200 Ilg/ml in sterile distilled water, dilute 1:100 in PNM
buffer.
- 20xSSC solution: dissolve 219.1 g sodium chloride and 110.3 g sodium
citrate in 1000 ml distilled water. Verify that the pH is between 6.5 and
7.5. Autoclave the solution for extended storage at room temperature.
- 2 x SSC solution: add 50 ml20 x SSC to 450 ml distilled sterile water and
adjust pH to 7.0 with 2 M HC!. Store at room temperature until solution
becomes cloudy.
- 2xSSC/0.1% Tween 20 buffer: add 0.1 % Tween 20 to 2xSSC
- 1xTE buffer: 10 mmol Tris HCI, 1 mmol EDTA, pH 8.0
- 4xT solution: add 25 ml Tween 20 to a solution of 100 ml20xSSC and
400 ml distilled sterile water and adjust pH to 7.5 with 2 N HC!. Store at
room temperature until the solution becomes cloudy.
413
- Hybridization Master Mix 2.1: 14.3% w/v dextran sulfate, 78.6% formamide, 2.9xSSC, pH 7.0. Add 1.45 mI20xSSC, 0.7 ml sterile distilled
water and 1.43 g dextran sulfate to 7.86 ml formamide. Heat to 70°C to
dissolve dextran sulfate. Aliquot in 1.5 ml tubes and store at -20°C.
- Mouse anti-FITC (DAKO), 430 Ilg/ml in sterile distilled water, dilute
1:200 in PNM buffer.
- 1x PBS/50 mM MgClz: add 50 mIlO x PBS and 25 ml 1 M MgClz to 425
ml sterile distilled water.
- IOxPBS (Dulbecco's PBS): dissolve 95.5 g PBS Dulbecco in 1000 ml
distilled water and sterilize the solution. Store at room temperature.
- Pepsin solution: prepare pepsin stock solution of 10 mg/ml in sterile
distilled water. Add 10-50 III stock solution to 100 ml 0.01 M HC!. Prewarm at 37°C in water bath, prepare fresh daily.
- PN buffer: (Na2HP04-7 hydrate/NaH 2 P0 4 -1 hydrate, pH 8, 0.05% nonidet P-40)
- PNM buffer: 5% nonfat dry milk, 0.1% NaN 3 in PN buffer
- Proteinase K solution: prepare proteinase K stock solution of 10 mg/ml
in sterile distilled water. Add 3 III stock solution to a buffer containing
2 mIl M Tris/HCI pH 7.5,0.2 mIl M CaClz and 97.8 ml distilled water.
Prewarm at 37°C in water bath, prepare fresh daily.
- Rhodamine-labeled sheep anti-digoxigenin (Roche Molecular Biochemicals), 200 Ilg/ml in sterile distilled water, dilute 1:100 in PNM
buffer.
- 20xSSC solution: dissolve 219.1 g sodium chloride and 110.3 g sodium
citrate in 1000 ml distilled water. Verify that the pH is between 6.5 and
7.5. Autoclave the solution for extended storage at room temperature.
- 2 x SSC solution: add 50 ml20 x SSC to 450 ml distilled sterile water and
adjust pH to 7.0 with 2 M HC!. Store at room temperature until solution
becomes cloudy.
- 2xSSC/0.1% Tween 20 buffer: add 0.1 % Tween 20 to 2xSSC
- 1xTE buffer: 10 mmol Tris HCI, 1 mmol EDTA, pH 8.0
- 4xT solution: add 25 ml Tween 20 to a solution of 100 ml20xSSC and
400 ml distilled sterile water and adjust pH to 7.5 with 2 N HC!. Store at
room temperature until the solution becomes cloudy.