104
TAKASHI SAWAI AND MIWA UZUKI
dNTP
(cold deoxynudeotide phosphate mixture)
- 0.3 mM dATP, dGTP, dCTP (or dTTP, if hot dCTP is used)
DNase I
- Stock solution: 1mg/ml DNase I (Boehringer) in 0.15 M
NaCl and 50% glycerol. Divide into small aliquots and
store at -20°C
- Working solution: (l:750 (mg/ml» To 1.5 ml of 0.9%
NaCl, add 2 III of stock DNase solution. Mix very gently.
Store on ice until use.
4. Incubate at 14°C for 2 hrs (Box made of styrofoam keeps
temperature constant for 2 hrs)
5. Stop reaction by adding 5 III of 0.2M EDTA, pH 8.0 and mix
6. Separate DNA using Nensorb 20 cartridge according to procedures suggested by the manufacturer.
Note:Washsamplewith3mlTris-TEA-EDTAandwithH201mlx2
7. Eluate with 1 ml of 50% methanol
8. Collect first 20 drops (ca. 500 ml)
9. Count 2 III by Scintillation counter (ideal specific activity of
labeled probe ( 3H: > lX10 7 , 35S: > lOB cpm Illg DNA)
10. Add ssDNA (lmg/ml): 8111 for 11lg cDNA (Final cone. 80 Ilg/
ml hybridization medium)
11. Divide into 10 aliquots and lyophilize
12. Store at -20°e.
Preparation of paraffin blocks for in situ hybridization
1. Fix tissues (lOmm x lOmm x 3mm) in 4% paraformaldehyde
in PBS for 1 hr at room temperature
2. Wash in three changes of PBS for 5 mins each
3. Dehydrate in a graded series of ethanol (70, 80, 90, 95, 100,
100 %), each for 30 mins
4. Clear in xylene (30 mins x 3)
5. Embed in paraffin
TAKASHI SAWAI AND MIWA UZUKI
dNTP
(cold deoxynudeotide phosphate mixture)
- 0.3 mM dATP, dGTP, dCTP (or dTTP, if hot dCTP is used)
DNase I
- Stock solution: 1mg/ml DNase I (Boehringer) in 0.15 M
NaCl and 50% glycerol. Divide into small aliquots and
store at -20°C
- Working solution: (l:750 (mg/ml» To 1.5 ml of 0.9%
NaCl, add 2 III of stock DNase solution. Mix very gently.
Store on ice until use.
4. Incubate at 14°C for 2 hrs (Box made of styrofoam keeps
temperature constant for 2 hrs)
5. Stop reaction by adding 5 III of 0.2M EDTA, pH 8.0 and mix
6. Separate DNA using Nensorb 20 cartridge according to procedures suggested by the manufacturer.
Note:Washsamplewith3mlTris-TEA-EDTAandwithH201mlx2
7. Eluate with 1 ml of 50% methanol
8. Collect first 20 drops (ca. 500 ml)
9. Count 2 III by Scintillation counter (ideal specific activity of
labeled probe ( 3H: > lX10 7 , 35S: > lOB cpm Illg DNA)
10. Add ssDNA (lmg/ml): 8111 for 11lg cDNA (Final cone. 80 Ilg/
ml hybridization medium)
11. Divide into 10 aliquots and lyophilize
12. Store at -20°e.
Preparation of paraffin blocks for in situ hybridization
1. Fix tissues (lOmm x lOmm x 3mm) in 4% paraformaldehyde
in PBS for 1 hr at room temperature
2. Wash in three changes of PBS for 5 mins each
3. Dehydrate in a graded series of ethanol (70, 80, 90, 95, 100,
100 %), each for 30 mins
4. Clear in xylene (30 mins x 3)
5. Embed in paraffin