232
HEINZ-ULRICH G. WEIER
- Cell fixative: acetic acid/methanol, 1:3 (vol:vol.). Make fresh before use.
- DB 0.5 solution: 0.5 M EDTA (pH 8.0), 1.0% N-Iauroyl sarcosine (Sigma), 0.5 mg/ml Proteinase K (Roche Molecular Biochemicals).
- Denaturing solution: 70% FA, 2xSSC, pH 7.0. Prepare fresh at least
every 2 weeks. Store at 4°e.
- ES Buffer: 0.5 M EDTA (pH 8.0), 1% sarcosyl.
- Gel loading dye: 1% bromophenol blue in 30% glycerol.
- Hybridization Master Mix: 14.3% w/vol dextran sulfate, 78.6% FA,
2.9xSSC, pH 7.0. For 10 ml MM2.1, mix 1,45 ml of 20xSSC with
0.7 ml ultrapure water, dissolve 1.43 g dextran sulfate (Calbiochem),
incubate overnight, then add 7.86 ml formamide. Aliquot in 1.5 ml microcentrifuge tubes and store at -20°e.
- Lysis buffer: 1% Triton X-lOO, 20 mM TrisHC1, 2 mM EDTA, pH 8.5.
- Maleic acid buffer: 100 mM maleic acid, 150 mM NaCl, adjust to pH 7.5
with concentrated NaOH
- Modified nucleotide mix (lOx) for labeling in combination with 1 mM
dig-l1-dUTP or FITC-12-dUTP: combine 5 III each of 100 mM dATP,
100 mM dGTP and 100 mM dCTP with 2.5 III of 1M Tris-HCL, pH 7.5,
0.5 III 0.5 M EDTA, pH 8.0 (Life Technologies) and 232 III ultrapure
water for a total of 250 Ill. Store at -20°e. The final concentration
of nucleoside triphosphates is 2 mM each.
- PNM: Dissolve 5 g of non-fat dry milk in 100 ml ofPN buffer (PN buffer
is 0.1 M sodium phosphate, pH 8.0, 0.1% nonidet-P40), incubate at
50°C overnight and add 1/50 vol. sodium azide, spin at 1000 g for
30 min, aliquot clear supernatant into 1.5-ml tubes and store at
4°e. Spin at 2000 g for 30 s prior to use.
- SCE: 1 M sorbitol, 0.1 M Na citrate, 10 mM EDTA, pH 7.8.
- Slide Blocking Solution (5 x SSC containing 2% Blocking Reagent, 0.1%
N-lauroyl sarcosine): combine 0.05 g N-lauroyl sarcosine (Na salt, Sigma) and 1 g Blocking Reagent with 12.5 ml of20xSSC (pH 7.0), add 30
ml water, heat to 60°C while stirring and bring the final volume to 50 ml
with ultrapure water, when the Blocking Reagent is dissolved. Aliquot
into 1.5-ml tubes, spin at 2000 rpm for 10 min and store at 4°e.
- SSC: 20xSSC is 3 M NaCl, 0.3 M Na3citrate x 2H 2 0, pH 7.0.
HEINZ-ULRICH G. WEIER
- Cell fixative: acetic acid/methanol, 1:3 (vol:vol.). Make fresh before use.
- DB 0.5 solution: 0.5 M EDTA (pH 8.0), 1.0% N-Iauroyl sarcosine (Sigma), 0.5 mg/ml Proteinase K (Roche Molecular Biochemicals).
- Denaturing solution: 70% FA, 2xSSC, pH 7.0. Prepare fresh at least
every 2 weeks. Store at 4°e.
- ES Buffer: 0.5 M EDTA (pH 8.0), 1% sarcosyl.
- Gel loading dye: 1% bromophenol blue in 30% glycerol.
- Hybridization Master Mix: 14.3% w/vol dextran sulfate, 78.6% FA,
2.9xSSC, pH 7.0. For 10 ml MM2.1, mix 1,45 ml of 20xSSC with
0.7 ml ultrapure water, dissolve 1.43 g dextran sulfate (Calbiochem),
incubate overnight, then add 7.86 ml formamide. Aliquot in 1.5 ml microcentrifuge tubes and store at -20°e.
- Lysis buffer: 1% Triton X-lOO, 20 mM TrisHC1, 2 mM EDTA, pH 8.5.
- Maleic acid buffer: 100 mM maleic acid, 150 mM NaCl, adjust to pH 7.5
with concentrated NaOH
- Modified nucleotide mix (lOx) for labeling in combination with 1 mM
dig-l1-dUTP or FITC-12-dUTP: combine 5 III each of 100 mM dATP,
100 mM dGTP and 100 mM dCTP with 2.5 III of 1M Tris-HCL, pH 7.5,
0.5 III 0.5 M EDTA, pH 8.0 (Life Technologies) and 232 III ultrapure
water for a total of 250 Ill. Store at -20°e. The final concentration
of nucleoside triphosphates is 2 mM each.
- PNM: Dissolve 5 g of non-fat dry milk in 100 ml ofPN buffer (PN buffer
is 0.1 M sodium phosphate, pH 8.0, 0.1% nonidet-P40), incubate at
50°C overnight and add 1/50 vol. sodium azide, spin at 1000 g for
30 min, aliquot clear supernatant into 1.5-ml tubes and store at
4°e. Spin at 2000 g for 30 s prior to use.
- SCE: 1 M sorbitol, 0.1 M Na citrate, 10 mM EDTA, pH 7.8.
- Slide Blocking Solution (5 x SSC containing 2% Blocking Reagent, 0.1%
N-lauroyl sarcosine): combine 0.05 g N-lauroyl sarcosine (Na salt, Sigma) and 1 g Blocking Reagent with 12.5 ml of20xSSC (pH 7.0), add 30
ml water, heat to 60°C while stirring and bring the final volume to 50 ml
with ultrapure water, when the Blocking Reagent is dissolved. Aliquot
into 1.5-ml tubes, spin at 2000 rpm for 10 min and store at 4°e.
- SSC: 20xSSC is 3 M NaCl, 0.3 M Na3citrate x 2H 2 0, pH 7.0.