27 Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells
331
- Bacto agar (Difco)
- Bacto peptone (Difco)
- Biotinylated goat-anti-avidin antiserum (Vector Labs)
- BSA (fraction V; Serva)
- BT buffer (0.15M NaHC0 3 , 0.1% Tween-20, pH 8.3). After dissolution
of NaHC0 3 , pH will be in the appropriate range. Make up fresh BT for
each experiment.
- Carrier DNA from salmon sperm or E. coli (Sigma). Dissolve at 10 mgt
ml in distilled (d)H 2 0 and shear by sonication to 100 bp-l kbp.
- DAPI (4,6-diamidine-2-phenylindole dihydrochloride; Sigma). Stock
solution: 1 mg/ml in sterile dH 2 0, store at -20°C.
- D-Glucose (Sigma)
- Dithiothreitol, make up 0.5 M stock with dH 2 0 and store DTT stock at
-70°C.
- DNA counterstains: The fluorescent dyes DAPI and PI are added to the
antifade solution in the following concentrations: DAPI 0.5 Ilg/ml and/
or PI 1 Ilg/ml.
- Ethanol (p.a.), ethanol (70%)
- Fixative: 3.7% acid-free formaldehyde, 4% sucrose, pH 7.4
- Formaldehyde 37%, acid free (Merck, Darmstadt)
- Hybridization solution: 50% deionized formamide, 10% 20xSSC, 20%
of a 50% (w/v) dextran sulfate solution, 10% of 50 mM sodium phosphate, pH 7.0, probe DNA at 30 ng/IlI, sheared herring carrier DNA at a
final conc. of 1 Ilg/Ill. Use deionized water to adjust volume.
- Lipsol. Lab cleaning reagent from LIP Ltd., Shipley, UK
- PBS, lOx: 1.3 M NaCI, 0.07 M Na2HP04, 0.03 M NaH 2 P0 4 , pH 7.4
- Pre-sporulation medium: 1% yeast extract, 2% Bacto peptone, 1% potassium acetate, pH 7.0. Make up fresh for each use.
- Propidium iodide (PI; Sigma). Dissolve at 1 mg/ml in sterile water,
store stock at -20°C.
- RNaseA (Sigma). Stock solution: 10 mg/ml RNaseA in dH 2 0, heat for
10 min to 95°C, store at -20°C.
331
- Bacto agar (Difco)
- Bacto peptone (Difco)
- Biotinylated goat-anti-avidin antiserum (Vector Labs)
- BSA (fraction V; Serva)
- BT buffer (0.15M NaHC0 3 , 0.1% Tween-20, pH 8.3). After dissolution
of NaHC0 3 , pH will be in the appropriate range. Make up fresh BT for
each experiment.
- Carrier DNA from salmon sperm or E. coli (Sigma). Dissolve at 10 mgt
ml in distilled (d)H 2 0 and shear by sonication to 100 bp-l kbp.
- DAPI (4,6-diamidine-2-phenylindole dihydrochloride; Sigma). Stock
solution: 1 mg/ml in sterile dH 2 0, store at -20°C.
- D-Glucose (Sigma)
- Dithiothreitol, make up 0.5 M stock with dH 2 0 and store DTT stock at
-70°C.
- DNA counterstains: The fluorescent dyes DAPI and PI are added to the
antifade solution in the following concentrations: DAPI 0.5 Ilg/ml and/
or PI 1 Ilg/ml.
- Ethanol (p.a.), ethanol (70%)
- Fixative: 3.7% acid-free formaldehyde, 4% sucrose, pH 7.4
- Formaldehyde 37%, acid free (Merck, Darmstadt)
- Hybridization solution: 50% deionized formamide, 10% 20xSSC, 20%
of a 50% (w/v) dextran sulfate solution, 10% of 50 mM sodium phosphate, pH 7.0, probe DNA at 30 ng/IlI, sheared herring carrier DNA at a
final conc. of 1 Ilg/Ill. Use deionized water to adjust volume.
- Lipsol. Lab cleaning reagent from LIP Ltd., Shipley, UK
- PBS, lOx: 1.3 M NaCI, 0.07 M Na2HP04, 0.03 M NaH 2 P0 4 , pH 7.4
- Pre-sporulation medium: 1% yeast extract, 2% Bacto peptone, 1% potassium acetate, pH 7.0. Make up fresh for each use.
- Propidium iodide (PI; Sigma). Dissolve at 1 mg/ml in sterile water,
store stock at -20°C.
- RNaseA (Sigma). Stock solution: 10 mg/ml RNaseA in dH 2 0, heat for
10 min to 95°C, store at -20°C.