19 cDNA Library Screening
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3. Typically dilutions in the range of 1 o- 3 to 1 o-s of this phage suspension are
retransformed into the bacterial host strain and rescreened at lower plaque density for positive signals either with radiolabeled probes or antibodies.
4. Steps 1. to 3. have tobe repeated until positive plaques can be punched
out of the top agar as single plaques derived from one single original
phage infection.
Phage propagation
1. To produce homogeneaus phage suspensions ofhigh titer, 100 f.ll to 250
f.ll of the individual plaque phage suspensions are plated in E. coli Y1088
for amplification.
Note: CHC1 3 must be removed from phage suspensions when plating dilutions less than w- 2 , since growth of host bacteria is severely inhibited by
traces of CHC13• To remove CHCh, blow a gentle stream ofN2 into the phage
suspension.
2. After overnight incubation at 42°C, one confluently lysed plate per phage
clone is overlaid with 7 ml A-dil, and phage particles are extracted by
constant shaking for 5 hrs at room temperature.
3. Supernatants containing phage are removed with a sterile 10 ml pipette
while carefully avoiding cellular debris. A few drops of CHC13 are added,
and the supernatants arestoredas phage stock solutions at 4°C indefinitely.
4. Phage stock titers should be in the range of 10 9 to 10 10 phage particles per
ml suspension. If a phage stock solution does not contain enough phage,
steps 1 to 3 are repeated until sufficient phage titers are achieved.
5. The homogeneity of the phage stocks should be tested by replating a
highly diluted aliquot of the stock (several hundred phages at a maximum) and rescreening with the probe. At least 99% of the tested plaques
should be positive to consider the tested phage stock "homogeneous".
305
3. Typically dilutions in the range of 1 o- 3 to 1 o-s of this phage suspension are
retransformed into the bacterial host strain and rescreened at lower plaque density for positive signals either with radiolabeled probes or antibodies.
4. Steps 1. to 3. have tobe repeated until positive plaques can be punched
out of the top agar as single plaques derived from one single original
phage infection.
Phage propagation
1. To produce homogeneaus phage suspensions ofhigh titer, 100 f.ll to 250
f.ll of the individual plaque phage suspensions are plated in E. coli Y1088
for amplification.
Note: CHC1 3 must be removed from phage suspensions when plating dilutions less than w- 2 , since growth of host bacteria is severely inhibited by
traces of CHC13• To remove CHCh, blow a gentle stream ofN2 into the phage
suspension.
2. After overnight incubation at 42°C, one confluently lysed plate per phage
clone is overlaid with 7 ml A-dil, and phage particles are extracted by
constant shaking for 5 hrs at room temperature.
3. Supernatants containing phage are removed with a sterile 10 ml pipette
while carefully avoiding cellular debris. A few drops of CHC13 are added,
and the supernatants arestoredas phage stock solutions at 4°C indefinitely.
4. Phage stock titers should be in the range of 10 9 to 10 10 phage particles per
ml suspension. If a phage stock solution does not contain enough phage,
steps 1 to 3 are repeated until sufficient phage titers are achieved.
5. The homogeneity of the phage stocks should be tested by replating a
highly diluted aliquot of the stock (several hundred phages at a maximum) and rescreening with the probe. At least 99% of the tested plaques
should be positive to consider the tested phage stock "homogeneous".