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Example sentences for: denhardt
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The rat VEGF probe is a 0.9 kb cDNA Pst I/Sma I insert cloned into a pBluescript II KS+ vector [ 33 ] . Prehybridization and hybridization were performed in 50% formamide, 5×SSC (20×SSC is 0.3 M sodium chloride, 0.3 < sodium citrate), 10× Denhardt's solution (50× Denhardt's solution is 2% Ficoll, 2% polyvinyl pyrrolidone, 2% Bovine Serum Albumin Factor V), 50 mM sodium phophate (pH 7.0), 1% sodium dodecyl sulfate (SDS), and 250 μg/ml salmon sperm DNA at 42°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.
The labeled DNA was mixed with 50 μl of 100 mM sodium acetate (pH 5.2), 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.
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.
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