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JOHANN PETER HOSSLE
Subprotocol 2
Hybridization Screening - cDNA Sequences as Probes
Materials
Solutions • Nitrocellulose membranes: Schleicher & Schüll {82 mm; 45 ~m)
• Denaturation solution: 0.5 M NaOH; 1.5 M NaCl
• Neutralization solution: 0.5 M Tris-Cl (pH 7.5); 1.5 M NaCl
• SSC: 3M NaCl; 0.3 M Na-citrate; autoclaved
• Sodium dodecyl sulfate (SDS): 20%
• Prewash solution: 50 mM Tris-Cl (pH 8.0); 1M NaCl; 1 mM EDTA; 0.1 o/o
SDS
• 20 x SSPE: 3M NaCl; 0.2 M NaH2P04; 25mM EDTA {pH 7.4); autoclaved
• 100 x Denhardt's solution: 2% Ficoll; 2o/o Polyvinylpyrrolidone; 2% BSA;
store at -20°C
• Calf thymus DNA {cT-DNA): 10 mglml; sonicated and stored at -20°C
• Hybridization solution: 5 x SSPE; 1 x Denhardt's; 100 ~g/ml cT-DNA;
0.1% SDS
• Radiolabeted ds cDNA probe: e.g. 32 P-labeled nick-translated cDNA
probe with a specific activity of 5 x 10 7 to 2 x 10 8 cpm/~g DNA
• X-ray film: Kodak XARS 35 x 43cm
• Whatman 3MM: filter paper
Procedure
1. Plate library as described above. For plaque hybridization experiments
use E. coli host strain Y1088.
2. Plaques develop at 42°C overnight.
3. Remove plates from the incubator and cool at 4°C for at least 1 hr.
4. Membrane lifts: First, the dry nitrocellulose membrane is marked with
the number of the plate that is to be replicated using a soft pencil.
JOHANN PETER HOSSLE
Subprotocol 2
Hybridization Screening - cDNA Sequences as Probes
Materials
Solutions • Nitrocellulose membranes: Schleicher & Schüll {82 mm; 45 ~m)
• Denaturation solution: 0.5 M NaOH; 1.5 M NaCl
• Neutralization solution: 0.5 M Tris-Cl (pH 7.5); 1.5 M NaCl
• SSC: 3M NaCl; 0.3 M Na-citrate; autoclaved
• Sodium dodecyl sulfate (SDS): 20%
• Prewash solution: 50 mM Tris-Cl (pH 8.0); 1M NaCl; 1 mM EDTA; 0.1 o/o
SDS
• 20 x SSPE: 3M NaCl; 0.2 M NaH2P04; 25mM EDTA {pH 7.4); autoclaved
• 100 x Denhardt's solution: 2% Ficoll; 2o/o Polyvinylpyrrolidone; 2% BSA;
store at -20°C
• Calf thymus DNA {cT-DNA): 10 mglml; sonicated and stored at -20°C
• Hybridization solution: 5 x SSPE; 1 x Denhardt's; 100 ~g/ml cT-DNA;
0.1% SDS
• Radiolabeted ds cDNA probe: e.g. 32 P-labeled nick-translated cDNA
probe with a specific activity of 5 x 10 7 to 2 x 10 8 cpm/~g DNA
• X-ray film: Kodak XARS 35 x 43cm
• Whatman 3MM: filter paper
Procedure
1. Plate library as described above. For plaque hybridization experiments
use E. coli host strain Y1088.
2. Plaques develop at 42°C overnight.
3. Remove plates from the incubator and cool at 4°C for at least 1 hr.
4. Membrane lifts: First, the dry nitrocellulose membrane is marked with
the number of the plate that is to be replicated using a soft pencil.