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Propagation and Processing of Phage
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes the procedure for propagating clones from the last step of affinity selection of phage. It is designed for the processing of up to approximately 80 clones simultaneously. The protocol consists of three sections. Section A (Single PEG-Precipitated Virions) is the basic protocol for obtaining phage to be used as sequencing templates. If the phage are to be used for ELISA, the methods in either Section B or Section C should be used.
Procedure
A. Single PEG-Precipitation of Virions
1. Inoculate each clone individually in 1.7 ml of NZY/Tet in 18 X 150 mm tubes.
2. Place the tubes vertically in a shaker-incubator at 37° C for approximately 16 to 24 hr (see Hint #2).
3. Pour each culture into a 1.5 ml microcentrifuge tube. Centrifuge samples briefly in a microcentrifuge at maximum speed to pellet the cells.
4. Pipette 1 ml of each supernatant into a fresh 1.5 ml microcentrifuge tube containing 150 μl of PEG/NaCl.
5. Mix by approximately 100 inversions and incubate at 4°C for at least 4 hr (see Hint #3).
6. Centrifuge in a microcentrifuge for 10 min at maximum speed to pellet the phage.
7. Remove the supernatant by aspiration.
8. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #5).
9. Remove any residual supernatant by pipetting or aspirating.
10. Dissolve the pellet in 20 μl of 0.1X TE by trituration with a pipette tip. The physical particle concentration is approximately 2.5 X 1013 virions per ml.
B. Double PEG-Precipitation of Virions
1. Inoculate each clone individually in 1.7 ml of NZY/Tet in 18 X 150 mm tubes.
2. Place the tubes vertically in a shaker-incubator at 37° C for approximately 16 to 24 hr (see Hint #2).
3. Pour each culture into a 1.5 ml microcentrifuge tube. Centrifuge samples briefly in a microcentrifuge at maximum speed to pellet the cells.
4. Pipette 1 ml of each supernatant into a fresh 1.5 ml microcentrifuge tube containing150 μl of PEG/NaCl solution.
5. Mix by approximately 100 inversions and incubate at 4°C for at least 4 hr (see Hint #3).
6. Centrifuge in a microcentrifuge for 10 min at maximum speed.
7. Remove the supernatant by aspiration.
8. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #5).
9. Remove any residual supernatant by pipetting or aspirating.
10. Dissolve the pellet in 1 ml of TBS by vigorous vortexing.
11. Centrifuge in a microcentrifuge for 1 min at maximum speed to clear the insoluble matter.
12. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube containing 150 μl of PEG/NaCl solution.
13. Mix by approximately 100 inversions and incubate at 4°C for at least 4 hr (see Hint #3).
14. Microcentrifuge for 10 min in a microcentrifuge at maximum speed.
15. Remove the supernatant by aspiration.
16. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #11).
17. Remove any residual supernatant by pipetting or aspirating.
18. Dissolve the pellet in 20 μl of 0.1X TE by trituration with a pipette tip.
19. Centrifuge briefly in a microcentrifuge at maximum speed to remove any insoluble matter.
20. Transfer the supernatant to a 0.5 ml microcentrifuge tube. The physical particle concentration is approximately 2.5 X 1013 virions per ml.
C. Double PEG-Precipitation and Acid-Precipitation of Virions (see Hint #5)
1. Inoculate each clone individually in 1.7 ml of NZY/Tet in 18 X 150 mm tubes.
2. Place the tubes vertically in a shaker-incubator at 37° C for approximately 16 to 24 hr (see Hint #2).
3. Pour each culture into a 1.5 ml microcentrifuge tube. Centrifuge samples briefly in a microcentrifuge at maximum speed to pellet the cells.
4. Pipette 1 ml of each supernatant into a fresh 1.5 ml microcentrifuge tube containing150 μl of PEG/NaCl solution.
5. Mix by approximately 100 inversions and incubate at 4°C for a minimum 4 hr (see Hint #3).
6. Centrifuge in a microcentrifuge for 10 min at maximum speed.
7. Remove the supernatant by aspiration.
8. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #5).
9. Remove any residual supernatant by pipetting or aspirating.
10. Dissolve the pellet in 1 ml of TBS by vigorous vortexing.
11. Centrifuge in a microcentrifuge for 1 min at maximum speed to clear the insoluble matter (see Hint #4).
12. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube containing 150 μl of PEG/NaCl solution.
13. Mix by approximately 100 inversions and incubate at 4°C for at least 4 hr (see Hint #3).
14. Centrifuge for 10 min in a microcentrifuge at maximum speed.
15. Remove the supernatant by aspiration.
16. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #11).
17. Remove any residual supernatant by pipetting or aspirating.
18. Dissolve the pellet in 90 μl of Unbuffered NaCl.
19. Centrifuge for 1 min in a microcentrifuge at maximum speed.
20. Transfer the supernatant to a 0.5 ml microcentrifuge tube containing 10 μl of 1 M Acetic Acid.
21. Mix by vortexing. Incubate for 10 min at room temperature.
22. Incubate for 10 min on ice.
23. Centrifuge for 30 min in a microcentrifuge at maximum speed at 4°C (see Hint #6).
24. Remove the supernatant by aspiration.
25. Centrifuge the samples again briefly (orienting the tube in the same direction in the centrifuge as the centrifugation in step #23).
26. Remove any residual supernatant by careful pipetting or aspiration as the pellet may be difficult to see.
27. Dissolve the pellet in 20 μl of 0.1X TE by trituration with a pipette tip.
28. Centrifuge briefly in a microcentrifuge at maximum speed to remove any insoluble matter.
29. Transfer the supernatant to a 0.5 ml microcentrifuge tube. The physical particle concentration is approximately 2.5 X 1013 virions per ml.
Solutions
Unbuffered NaCl
0.15 M NaCl
TE (1X)
Autoclave and store at room temperature
Adjust the pH to 8.0
1 mM EDTA
10 mM Tris-HCl
NZY/Tet (1X)
Prepare in NZY Medium
20 μg/ml Tetracycline
NZY Medium (1X)
Also see Hint #1
10 g NZ amine A
5 g NaCl
Store at room temperature
Prepare in 1 liter of ddH2O
Autoclave
Adjust the pH to 7.5 with NaOH
5 g Yeast Extract
1 M Acetic Acid
BioReagents and Chemicals
Acetic Acid
Sodium Hydroxide
Tris-HCl
Yeast Extract
NZ Amine A
Tetracycline
EDTA
Sodium Chloride
Protocol Hints
1. The contributors of this protocol routinely use NZY. Other media such as LB can also be used. NZY has the advantage that NZ Amine A (from Humko Sheffield Chemical, P.O. Box 630, Norwich, NY 13815) is much cheaper than tryptone.
2. 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.
3. Incubation overnight gives a higher yield.
4. The next steps (Steps 12 to 14; a second PEG precipitation with clearing to remove insoluble matter) are optional, but are recommended if the phage are to be used for ELISA assay. They are unnecessary if phage are to be used as sequencing templates.
5. 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.
6. As the pellet may be difficult to see or invisible, note the orientation of the tube in the microcentrifuge. This will allow localization of the pellet--at the point of maximal centrifugal force during the centrifugation steps.
