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Propagation and Processing of Phage on the 1.7-ml Scale
NOTE: This is the routine procedure for propagating clones from the last step of affinity
selection. It is designed for processing up to ~80 clones simultaneously. Assuming all clones
have the same provenance, it is unnecessary to label individual tubes until the very last step. The
culture tubes used for propagation in step 1 should be sterile; the Ep tubes and tips can be used
right out of the bag.
PEG PRECIPITATION
1. Inoculate 1.7-ml of NZY containing 20 g/ml tetracycline in 18 × 150 mm tubes with clones;
shake the tubes vertically in a shaker-incubator at 37º for ~16-24 hr.
NOTE: Vertical shaking does not aerate the culture enough to support aerobic growth in the late
stages of the growth curve. Nevertheless, the yield of fd-tet-based phage is not greatly affected
by oxygen deficit. Perhaps, because of the replication defect in these phage, it is phage DNA
replication, not cellular growth, that limits phage production.
2. Pour each culture into a 1.5-ml microcentrifuge tube. Microfuge briefly to pellet cells.
3. Pipette 1 ml of supernatant into a second 1.5-ml microcentrifuge tube to which 150 l
PEG/NaCl solution has already been added. Mix by ~100 inversions and incubate in refrigerator
for at least 4 hr (overnight better).
4. Microfuge 10 min; RRR.
NOTE: The next two steps (a second PEG precipitation with clearing to remove insoluble
matter) are optional, but probably advisable if the phage are to be used for ELISA. They
are unnecessary if the phage are simply to be used as sequencing templates.
5. Dissolve pellet in 1 ml TBS by vigorous vortexing. Microfuge 1 min to clear insoluble
matter; transfer the supernatant to a fresh 1.5-ml microcentrifuge tube to which 150 l
PEG/NaCl solution has already been added. Mix by ~100 inversions and incubate in
refrigerator for at least 4 hr (overnight better).
6. Microfuge 10 min; RRR.
7. Dissolve the pellet in 20 µl 1/10 × TE by scraping and pumping briefly with the pipette tip
and vortexing. The physical particle concentration is ~2.5 × 1013 virions/ml.
NOTE: The next step (clearing to remove insoluble matter) is optional, but advisable if
phage are to be used for ELISA. It is unnecessary if the phage are simply to be used as
sequencing templates.
8. Microfuge 1 min; transfer supernatant to a 500-µl Ep tube.
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ACID PRECIPITATION
NOTE: Filamentous phage precipitate isoelectrically in 0.1 M acetic acid; this can be used as an extra
dimension of purification when particularly pure phage are needed. We seldom exercise this option.
The following alterations are made to steps 7 and 8 above:
7´. Dissolve the pellet in 90 µl unbuffered 0.15 M NaCl (instead of 20 µl 10 mM Tris.HCl pH
8).
8´. Microfuge 1 min; transfer supernatant to a 500-µl Ep tube that aleady contains 10 µl 1 M
acetic acid. Mix by vortexing; incubate 10 min at room temperature and 10 min on ice.
9. Microfuge 30 min in cold, being sure to orient tube in the microfuge so that you’ll know which is the
centrifugal edge (pellet may not be visible); RRR, being careful to avoid the (possibly invisible) pellet.
Dissolve pellet in 20 l 1/10 × TE.
10. Clear insoluble matter by microfuging ~1 min; transfer the supernatant to a fresh 500-l Ep
tube. The physical particle concentration is ~2.5 × 1013 virions/ml.