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Large-Scale Purification of Wild-Type Virions With or Without Detergent Treatment
NOTE: This protocol is adapted from Wickner, W., Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli , Proc. Natl. Acad. Sci. USA 72 , 4749–
4753, 1975. It yields highly purified phage without loss of infectivity, presumably by desorbing traces of adherent proteins. However, that finding by no means precludes the possibility of subtle alteration of virion structure. For cases where detergent treatment is inadvisable we also show in boxes the modifications required if you want to omit detergent treatment.
NOTE: Phage fd-tet and its derivatives have no minus-strand origin, and therefore give low intracellular RF copy numbers and reduced virion yield (~5 × 10
11
virions/ml). Wild-type phage
(including Messing's M13mp series) give ~3 × 10 12
virions/ml under the conditions outlined below. This protocol is essentially the same as that for fd-tet-based phage
( VirionPurification.doc
), but scaled up and modified to accommodate the different mode of propagation (here we usually start with a plaque rather than a tetracycline-resistant colony) and the higher yield of phage.
MATERIALS
Inoculum of wild-type virions: ~2 × 10
11
virions in up to ~200 µl. Ten-fold errors on either side shouldn’t matter. A suitable inoculum can be prepared from a single plaque as follows: a. Inoculate 5 ml NZY (or LB
) in a 1 × 3.5 inch culture tube (or other suitable sterile vessel) with 50
 l of an overnight culture of K91BluKan grown in the same medium (100
 g/ml kanamycin optional). Shake the culture until turbidity just becomes evident. b. Inoculate the culture with a single plaque, using a sterile inoculating loop or a sterile wooden stick. c. Shake at 37º overnight. d. Use 50 µl (nominally ~1.5 × 10 11
virions) as the inoculum.
Four 3.8-liter fernbach flasks with 1 liter NZY prewarmed to 37° in the shaker-incubator
The shaker-incubator will also have to have a clip for a 250-ml side-arm flask
36 500-ml centrifuge bottles for Sorvall GS3 rotor
PROCEDURE
1. In a 250-ml side-arm flask inoculate 25 ml NZY with 250 µl of an overnight culture of
K91BluKan. Shake vigorously at 37° until the OD
600
= ~0.2.
2. Use 2.5 ml to inoculate each of the four fernbach flasks; then infect the remaining 15 ml in the 250-ml side-arm flask with ~2 × 10
11
wild-type virions in 200 µl or less. Continue to shake

726932913 Page 2 4/15/2020 all five flasks (four fernbach flasks and the 250-ml side-arm flask) until the OD
600
in the sidearm flask reaches 0.6.
3. Pour the infected culture in the 250-ml side-arm flask into a disposable 50-ml conical centrifuge tube, and pipette 3 ml into each of the four fernbach flasks. Continue to shake the
Fernbach flasks vigorously overnight at 37°.
Here is a typical time-course for steps 1–3:
Elapsed time (hr) OD
600
Operation
0 Inoculate 25-ml culture with cells (step 1)
2:41 0.2
2:55
0.41
0.5
Inoculate 1-liter cultures with 25-ml culture, and 25ml culture with fd (step 2)
3:22
3:52
4:26 0.6 Inoculate 1-liter cultures with fd (step 3)
4. Clear by two centrifugations in 500 ml bottles at 5 and 8 Krpm 10 min. Pour the final supernatants into tared 500-ml bottles (12 bottles altogether for 4 liters).
5. To each bottle add 0.15 volume PEG/NaCl . Allow to precipitate overnight in cold.
6. Centrifuge bottles 8 Krpm 20 min; RRR ; to each bottle add 10 ml TBS ; shake bottles in the cold (room temperature OK) to resuspend phage. Pool all the phage (120 ml altogether; call this
“
1 vol
”) in a single 250-ml centrifuge bottle. Centrifuge at 12 Krpm 15 min in GSA rotor to pellet insoluble matter. Pour and pipette the supernatant into a fresh 250-ml centrifuge bottle.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: Add an additional 13.3 ml water to the supernatant and skip directly to step 8.
7. To the 250-ml bottle add 13.3 ml ( 1/9 vol ) of a 1:9 v/v mixture of Triton-X100:water. Shake gently in shaker incubator at room temperature for 1 hr.
8. Add 20 ml PEG/NaCl ( 1/6 vol ). Mix by many inversions. Centrifuge at 10 Krpm in the GSA rotor for 15 min. RRR . Dissolve pellet in 60 ml TBS by shaking bottle in shaker-incubator at room temperature. When the phage are dissolved, centrifuge at 10 Krpm in the GSA rotor for 10 min. Pour and pipette the supernatant into a fresh 250-ml bottle.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: Dissolve in 120 ml TBS rather than 60 ml, and skip directly to step 10.
9. Add 60 ml 2% sarkosyl in TBS and shake gently in shaker incubator at room temperature for
1 hr.

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10. Add 18 ml PEG/NaCl . Mix by many inversions. Centrifuge at 10 Krpm in the new GSA rotor for 15 min. RRR . Dissolve pellet by shaking bottle in the cold (room temperature OK).
11. Centrifuge at 10 Krpm 10 min in SLA1500 rotor to clear insoluble matter.
12. Into a tared 250-ml glass beaker (make sure it’s clean!) weigh 29.70 g CsCl. Tare the beaker again. Pour and pipette the cleared supernatant from the previous step into the beaker, plus sufficient extra TBS to bring the net weight of aqueous phase 66.11 g. Stir gently to dissolve the salt. You should now have 73.7 ml of a 31% w/w (1.30 g/ml) solution. Confirm that the density is 1.29–1.31 g/ml. (NOTE: Put the beaker into the non-disposable waste to be autoclaved.)
13. Divide equally into two 1 × 3.5 inch centrifuge tubes for the SW28 rotor. Centrifuge at 15° for 36–48 hr at 25,000 rpm. There should be a clean phage band toward middle of each tube; this band is quite translucent and can be difficult to see at first. Its upper boundary is very sharp, and is distinctly noticeable as a refractive index discontinuity even if light scattering from the phage band is very feeble.
FOR PURIFICATION WITHOUT DETERGENT TREATMENT: If detergent treatment is omitted, there will usually be a sharp, thin flocculent band above the phage band, and another much larger flocculent band just below the phage band. These flocculent bands are relatively opaque, and are therefore much more apparent at first glance than the very translucent phage band. We try to avoid the lower flocculent band if possible, but there’s little harm if some of this band contaminates the phage band.
14. Using a sterile transfer pipette, remove the solution above the phage bands, trying as much as possible to keep the tip at the meniscus; then use a fresh transfer pipette to remove the phage bands and pool them into a single 50-ml disposable conical centrifuge tube. Bring the total volume in the 50-ml tube to 50 ml by adding additional TBS .
15. Dialyze in two Pierce 30-ml Slide-A-Lyzers (or other form of dialysis membrane) against three changes of ~2 liter of TBS .
16. Remove the dialysates into a single suitable vessel and store in the refrigerator.
17. In a sterile 1.5-ml Ep tube dilute 5 µl of the phage prep with 495 µl water. This is 1/100 dilution. Scan from 220–320 nm, using water as the blank; discard the scanned solution but save the rest of the 1/100 dilution. Calculate the actual undiluted physical particle concentration as in
AbsorptionSpectrum.doc
. A typical phage concentration would be ~2 × 10 14 virions/ml.
18. Titer suitable dilutions of the 1/100 dilution previous step for plaque forming units (pfu) as in Plaques.doc
. The infectivity (pfu/virion) will typically be ~50%.