306
JOHANN PETER HOSSLE
Subprotocol 4
Purification of Phage Particles, DNA Isolation and Subcloning
Materials
• SM (without gelatin): 0.1 M NaCl; 8 mM MgS04; 50 mM Tris-Cl (pH 7.5);
autoclaved
• RNase A: 1 mg/ml in H20
• DNase 1: 1 mg/ml in H20
• CsCl: Solutions of different densities:p=l.45, 1.5, and 1.7
• Solutions for balancing: 5 ml SM + 2.5 g CsCl, 5 ml SM + 3.75 g CsCl
• Refractometer
• Polyethylene glycol (PEG): 6000 (SERVA)
Procedure
I. Foreach ofthe phage tobe prepared, 10 LB plates with at least 10 5 phage
are plated in E. coli Y1088 and incubated at 42°C overnight.
2. Supernatants containing phage are prepared as described previously,
and the supernatants from all 10 plates (10 x 5 ml) are pooled.
3. Each pool is divided into two 25 ml aliquots in 40 ml polypropylene centrifuge tubes.
4. Bacterial nucleic acid is digested completely by addition of25Jll RNase A
and DNase I, then incubation at room temperature for 30 min.
5. 1.46 g NaCl is added per tube and dissolved completely. After 1 hr at
room temperature, cell debris is removed by centrifugation at 10,000
rpm for 10 minutes at 4°C.
6. Supernatants are transferred into a fresh 40 ml polypropylene tube and
2.5 g PEG is added and completely dissolved by constant shaking for 30
min at room temperature. Phage particles are PEG-precipitated overnight on ice.
7. Phage precipitates are recovered by centrifugation at 10,000 rpm for 10
min at 4°C.