Denhardt s solution

X Denhardt s solution 1% (w/v) Ficoll, 1% (w/v) polyvinyl pyrrolidone, 1% (w/v) BSA Filter-stenlize Store aliquots at -20°C. Do not flame the pipets used to transfer this solution. Denaturation and precipitation of the protein result from the use of hot pipets at this stage... 

Prehybndization solution- 50% formamide, 5X SSC, 5X Denhardt s solution, 25 xaM sodium phosphate, pH 6.5,300 pg/mL fleshly denatured sheared herring sperm DNA Filter through Whatman no 1 paper on a Buchner funnel, then through a sterile 0 45-pm filter Store 10-mL aliquots m glass at-20°C Use only once... 

X Denhardt s solution- 0.2 g BSA, 0 2 g ficoll, 2 g glycine, 0.2 g polyvinyl pyrrolidone, sterile distilled water to 10 mL. Store m aliquots at—20°C... 

Prehybridization/hybridization buffer 50% deionized formamide, 5 X SSC (standard saline citrate), 8 X Denhardt s solution, 50 mM sodium phosphate buffer (pH 6.5), 0.5% sodium dodecyl sulfate (SDS), 250 fig/ml denatured herring testes DNA, and 500 ng/ml yeast RNA (for the composition of SSC buffer and Denhardt s solution, see, e.g., Ausubel et al.13)... 

Blots are prehybridized to block all nonspecific binding (which causes background). To do this, one first wets the nitrocellulose in 3 X SSC, then in 50% formamide, 3X SSC, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA (pH 8.0), 10 mM Tris (pH 7.5), 10X Denhardt s solution [1X Denhardt s is 0.02% Ficoll, 0.02% bovine serum albumin (BSA), and 0.02% polyvinylpyrrolidone 360], 0.05% sodium pyrophosphate, and 100 /ig/ml denatured DNA. Denatured DNA is sheared herring or salmon sperm DNA that has been boiled for 10 min before addition to the prehybridization mix. Blots are sealed within plastic bags, placed between two glass plates to give uniform distribution of prehybridization solution over the blot, and incubated in a water bath at 37° for at least 1 hr. 

Stir for 45 min. Filter and store at -20°C. lOOX Denhardt s solution 0.2 g of Ficoll, 0.2 g of polyvinyl pyrrolidone, 0.2 g of bovine serum albumin (BSA). Add sterile distilled water to 10 mL. Store aliquots at -20°C. 

Hybridization solution 45% Formamide, 5X SSC, 5X Denhardt s solution, 20 mMsodium phosphate, pH 6.5, 300 pg/mL freshly denatured, sheared, herring sperm DNA, 200 ng of biotinylated DNA/mL. Before its addition, the biotinylated probe DNA is denatured by incubating for 10 min in a boiling water bath and quickreduce size is unnecessary, since the products generated by nick translation are sufficiently small. Filter and store the hybridization solution as was done for the prehybridization solution. Hybridization solution can be recovered after use and stored at-20°C. The solution can be reused at least 10 times over a time-span of at least 5 mo, without noticeable... 

Denhardt s solution (2% polyvinylpyrolidone, 2% bovine serum albumin [Pentax , fraction V]). 

RNA, if not too small, can also be detected by in-gel hybridization. Instead of denaturation/neutralization, the formaldehyde-containing gel is washed for 30 min in 0.1 M Tris-HCI (pH 7.5) (Wallace and Miyada, 1987). Ahmad et al. (1990) fractionated RNA in a formaldehyde-containing agarose gel in MOPS buffer (Section 9.1.3), washed the gel in DEPC-treated water (twice) and placed it between two sheets of gel blot paper (S S GB002) before drying in a gel dryer as described in the previous section. They hybridized with cDNA at 42°C in 50% formamide, 5 X Denhardt s solution and 5 X SSPE as described in Section 9.3.2. 

Remove prehybridization solution without permitting the specimens to dry and add probe in hybridization solution (radiolabeled probes 10—600 ng/ml nonradioactive probes up to 10 times more). Hybridization solution contains 50% formamide, 0.5 M NaCl, 10 mM Tris-HCI (pH 7.6), 2 mM EDTA, 1 x Denhardt s solution, 20 mM DTT, 300 pg/ml tRNA, 300 (xg/ml poly(A) or/and poly(C) (optional), 10% PEG 6000 RNase-free. S label may form nonspecific disulfide bonds in the cell if reductants, such as DTT or 2-mercaptoethanol, are omitted. 

Staining solution 305pLofH20,150pLof20X SSPE, 10 pL of 50X Denhardt s solution, 5 pL of 1% Tween-20, 12.5 pL of 1 mg/mL SAPE (Molecular Probes, Eugene, OR), and 5 pL of 0.5 mg/mL Biotinylated anti-streptavidin (Vector Laboratories, Burlingame, CA). 

Hybridization buffer 50% deionized formamide, 5X SSC, 10% dextran sulfate, 5X Denhardt s solution, 2% SDS, and 100 Jg/mL denatured sheared herring sperm DNA. Make this solution fresh before use, and store at 50°C. 

Hybridization buffer 50% (v/v) deionized formamide, 0.3M NaCl, 20mM Tris-HCl, pH 8.0, 5mM EDTA, pH 8.0,10% (w/v) dextran sulfate, lx Denhardt s solution, 0.5 iglvoL yeast tRNA. Store in aliquots at -TO C. 

ISH buffer To 5 mL of 100% deionized formamide (Boehringer Mannheim), 1 mL of 20X SSC, 200 0,L of lOOX Denhardt s solution, 1 g of dextran sulfate, 4 mg of yeast tRNA, 2 5 mg of salmon-sperm DNA (predenatured), 200 pL of 1 M dithiothreitol (DTT), add DEPC-treated water to a final volume of 10 mL. Stir overnight, aliquot, and store at -80°C... 

Salts lOX stock solution 3 M NaCl, 200 itiAf Tris-HCl, pH 6.8, 50 mM EDTA, 10 mM sodium phosphate buffer pH 6 8, lOX Denhardt s solution, stored at-20°C. 

Washing solution. 50% formamide, IX salts, 10 mM DTT. lOX salts stock solution IS as described in Subheading 2.6. except that Denhardt s solution is omitted The formamide need not be deionized. 

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