Partial Purification of Virions from 7-Ml Cultures

SmallScaleVirions.doc 10/13/18 Page 1

Partial Purification of Virions From 7-ml Cultures

NOTE: This procedure yield partially purified virions suitable for binding assays (e.g., ELISA.doc) and other types of analysis. It assumes that cells harboring fd-tet-based display phage are available, either as colonies on plates or in some other form. Alternatively, culture supernatants from step 2RFmidiprep2.doc can be processed starting at step 3 below.

1. Use well separated colonies from the petri dishes (or some other source of phage-bearing cells) to inoculate 1 × 3.5 inch culture tubes (or other suitable culture vessel) containing 6 ml NZY + 0.2 µg/ml tetracycline[1]; shake the tubes vigorously ~2 hr at 37º; then pipette 1 ml NZY + 140 µg/ml tetracycline into each culture tube; continue shaking overnight at 37ºC.

2. Pour cultures into 50-ml tubes; centrifuge at 5 Krpm 10 min in the cold in the FiberLite rotor[2]; pour off supernatants into fresh 50-ml tubes.

3. Re-centrifuge the once-cleared supernatant previous step (or from step 2 of RFminiprep2.doc) for 10 min at 8 Krpm; pour the doubly-cleared supernatants into fresh 50-ml tubes.

4. To each tube add 1.05 ml PEG/NaCl; vortex; allow to stand in the cold overnight.

5. Centrifuge for 15 min at 14,000 rpm in the FiberLite rotor to pellet the virions; RRR; dissolve the pellets in 1 ml TBS.

6. Transfer the solutions to 1.5-ml Ep tubes; microfuge 1 min at top speed to pellet insoluble material; carefully transfer the supernatants to fresh 1.5-ml Ep tubes; to each Ep tube add 150 µl (nominally 0.15 vol) PEG/NaCl; vortex; allow to precipitate in the cold at least 4 hr (overnight better).

7. Microfuge for 10 min at top speed to pellet virions; RRR; dissolve the pellets in 70 µl TBS; microfuge 1 min at top speed to pellet insoluble material; carefully transfer the supernatants to fresh 500-µl Ep tubes; store these in the refrigerator. The expected physical particle concentration is ~5 × 1013 virions/ml, equivalent to and infectious unit titer of ~2.5 × 1012TU/ml.

[1] This concentration of tetracycline is sub-inhibitory but sufficient to induce expression of the tetA gene (the resistance protein). The 2-hr induction period before challenge with an inhibitory concentration of tetracycline ensures that a clone will be propagated even if it’s phenotypically sensitive at the time of inoculation. Usually this precaution isn’t necessary, since TetA mediated resistance is phenotypically unusually stable.

[2] These excellent rotors can centrifuge ordinary disposable 50-ml tubes at 15,000 rpm! If a FiberLite rotor isn’t available, you can use OakRidge tubes in the SS34 rotor or any other suitable rotor.