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Example sentences for: denhardt
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A rabbit genomic library in Lamda DASH ®II vector (Stratagene) was screened with [ 32P]-labeled full-length human EP 2 (gift from Dr. D. Woodward, Allergan Pharmaceuticals) [ 20 ] . The filters were fixed by ultraviolet light exposure (Stratalinker, Stratagene) and hybridized 18 hours at 56°C in a solution containing 6X SSC, 5X Denhardt's, 0.1% SDS, 150 μg/ml denatured herring sperm DNA and 3 × 10 6cpm/ml labeled probe.
Isolation of total cellular RNA using a urea/LiCl protocol and gel electrophoresis was performed as described previously [ 65 ] . Gels were transferred onto Amersham Hybond N+ nylon membranes in 20 × SSC and prehybridized in 4 × SSC, 1 × Denhardt's solution, 0.5% SDS for 2 h at 68°C.
The Cy3 (HL60) and Cy5 (HL60/TLK286) PCR products were purified with Qiagen PCR purification kit, reduced to a final volume of 7.5 μl with a speed vac, mixed with 3.75 μl of 20x SSC, 0.75 μl of 10% SDS, 1.5 μl of 1 μg/μl salmon DNA and 1.5 μl of 50x Denhardt's solution, denatured at 95°C for 5 min, cooled on ice and incubated at 42°C for 15 min.
Hybridization was carried out with the 35S-labeled RNA probe (4-6 × 10 6cpm/ml) in a solution containing 50% (vol/vol) deionized formamide, 0.3 M NaCl, 10 mM Tris (pH 8.2), 1 mM EDTA, 0.05% yeast tRNA, 10 mM dithiothreitol, 1 × Denhardt's solution and 10% dextran sulfate.
Briefly, filters were pre-hybridized at 65°C in hybridization buffer (5x SSC, 50 mM NaPO 4 , pH 7.4, 2.5x Denhardt's Solution) with 100 μg/ml denatured salmon sperm DNA for two hours.
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