CsCl phage preps - Center for Phage Technology

Protocol: Purifying phage by CsCl gradient ultracentrifugation
Materials required:
• Large volume (e.g., 4 liter) culture flasks and an appropriate incubator
• Visible-light spectrophotometer
• DNase I and RNase A, powder or stock solutions
• Polyethylene glycol 8000, NaCl, chloroform (for optional PEG precipitation)
• Large high-speed centrifuge with large capacity (e.g. GSA, GS-3 or JA10) and
small-capacity (e.g., SS-34) rotors, centrifuge bottles and tubes
• Gelatin-free SM buffer (100 mM NaCl, 25 mM Tris-HCl pH 7.5, 8 mM MgSO4)
• CsCl
• Ultracentrifuge, rotor and appropriate disposable tubes (tubes should be clear
and able to be punctured by a standard 20 ga. needle)
• 10 cc syringes, 18 – 20 ga. needles
• Dialysis cassettes or tubing, 3 kDa – 50 kDa NWCO cutoff
Lysate preparation
1) Use 1-2 liters of phage liquid culture, phage titer should be at least 1 x 109
PFU/ml for best results. Having too few phage will make the band in the CsCl
gradient difficult to find. In a 70.1 Ti quickseal tube, about 5 x 1010 PFU are
required to see a band.
a. Lysate propagation for virulent phage varies from phage to phage and
host to host. A basic starting protocol is to subculture your host into 1 or
2 liters of fresh broth medium (starting OD550 should be about 0.05) and
incubate until the culture reaches and OD550 equal to about 5 to 10% of
the host’s stationary phase OD550 (e.g., if a saturated culture of your host
has an OD550 of ~5, wait until the culture reaches and OD550 of about
~0.25). At this point, inoculate the culture with your phage so that the
final MOI is about 0.025, and monitor the culture growth at OD550. Ideally,
you should see the OD of the culture grow, plateau and then drop off.
When the culture OD stops dropping (it will probably not clear
completely), move on to the next step and harvest the lysate. If the lysate
titer is too low, you can try changing the point at which you inoculate the
culture, or changing the amount of phage you add.
2) Centrifuge the liquid culture at 12,000 x g, 15 min, 4 ºC.
3) Carefully decant the supernatant into a clean Erlenmeyer flask. The supernatant
should be optically clear (OD550 of about 0.05 or less); centrifuge again if it is not.
Filtration of the supernatant to remove cell debris (0.45 µm) is a good idea if
practical, although this is not strictly required.
4) Add DNase I and RNase A to the cleared lysate to a final concentration of 1
µg/ml each. Stir or gently shake the lysate at RT for 1-2 hr. Lysates usually can
be stored for up to 48 hrs at 4 ºC.
Center for Phage Technology
Texas A&M University, College Station, TX 77843
Protocol: CsCl phage prep
Rev. 08/18/2011
1 of 3
Phage concentration
You can concentrate the phage by one of two methods: high-speed centrifugation, or
precipitation in the presence of NaCl and polyethylene glycol. The centrifugation method
usually provides a cleaner prep with less chance for phage inactivation.
Centrifugation method
1) Tailed phages have sedimentation coefficients of about 300-1200; for example,
lambda is about 400 while T4 is about 800-1000. Calculate the pelleting time for
your phage based on its estimated size, using the formula t = k/S, where t is the
time in hours, k is the rotor k factor, and S is the sedimentation coefficient.
Centrifuge the phage in clean bottles at 4 ºC for a time equal to two to four times
the calculated pelleting time. A typical run for a phage like lambda would be
7,000 x g, 4 ºC for 16 – 18 h in a Sorvall GSA or GS-3 rotor.
2) Carefully pour off the supernatant and retain the pellet, which will be glassy or
opaque. Do not disturb the pellets.
3) Add enough gelatin-free SM buffer to just cover the pellets and let them soak at 4
ºC for a few hours to overnight, until they loosen up. When loose, gently mix to
resuspend thoroughly transfer to a 50 ml centrifuge tube. The final resuspension
volume should be about 5 - 10% of the original lysate volume.
4) Centrifuge the phage suspension again at 12,000 x g, 4 ºC for 10 min to pellet
any remaining cell debris. Transfer the supernatant to a new tube. Continue to
the CsCl gradient procedure.
PEG precipitation method
1) Add solid NaCl to the lysate to a final concentration of 1 M (58.4 g/l). Stir until
dissolved.
2) Add solid PEG-8000 to the lysate to a final concentration of 10% (100 g/l). Stir
until dissolved, then stir on ice for at least 30 min.
3) Place the lysate flask at 4 ºC (no stirring) overnight.
a. This is a convenient stopping step; some phages (e.g. BcepIL2, S. aureus
phage K) can be left in this state for at least several days.
4) Centrifuge the PEG-precipitated lysate at 10,000 x g, 10 min, 4 ºC.
5) Pour off the supernatant and let the centrifuge bottles stand inverted for 3-5 min
to drain any excess supernatant from the pelleted precipitate.
a. Examine the supernatant after pouring off to ensure most of the
precipitate remained in the pellets. Centrifuge again if a significant
portion of the pellets sloughed off into the supernatant.
6) Resuspend the PEG precipitate in a minimal volume of lambda-diluent (100 mM
NaCl, 10 mM MgSO4, 50 mM Tris-HCl, pH 7.5).
a. Start with about 18 ml of lambda-dil per 2 litres of starting lysate, use a
greater volume if necessary. Don’t vortex; swirling and gently pipetting
with a wide-bore 1 ml pipette tip helps.
7) Transfer the resuspended precipitate to a 50 ml Falcon tube, and centrifuge at
3,000 x g, RT, 10 minutes to pellet any insoluble material. Discard the pellet.
8) Transfer the supernatant to a clean polypropylene or teflon Oak Ridge tube. Add
an equal volume of chloroform and mix by inverting for 2 min.
a. Be sure to use a chloroform-compatible tube; clear polycarbonate is not
compatible with chloroform.
Center for Phage Technology
Texas A&M University, College Station, TX 77843
Protocol: CsCl phage prep
Rev. 08/18/2011
2 of 3
9) Centrifuge at 12,000 x g, 10 min, 4 ºC. Transfer the supernatant to a new Oak
Ridge tube; avoid the chloroform and the white interface layer.
10) Repeat steps 12 and 13 until there is little or no white precipitate at the interface.
Retain the phage suspension. Continue to the CsCl gradient procedure.
Isopycnic CsCl gradient centrifugation
1) Carefully measure the volume of the phage suspension and transfer it to a 50 ml
Falcon tube. Add solid CsCl at a rate of 0.75 g/ml of phage suspension. Add the
CsCl slowly (about ¼ at a time) and swirl gently to dissolve.
a. The final density of the solution should be about 1.40 – 1.45 g/ml.
2) Load the phage-CsCl solution to ultracentrifuge tubes and run.
a. Using a Beckman 70.1 Ti rotor: 45,000 RPM, 5 ºC, 24 hrs.
b. Using a Beckman SW28 rotor: 25,000 RPM, 5 ºC, 24 hrs.
c. If there is not enough phage suspension to completely fill the tubes, top
them up with a solution of gelatin-free SM buffer containing 0.75 g/ml
CsCl. Be sure to mix the phage and added buffer thoroughly as the
densities of the two solutions will probably not be identical.
d. Be sure to balance the tubes to within 10 mg of each other before loading
into the rotor.
3) The phage should form a grey-white band somewhere in the middle third of the
tube. Clamp the tube into a retort stand and carefully insert a 10 cc syringe with
an 18 or 20 ga. needle through the wall of the tube, 2-3 mm below the band.
Carefully draw out the band with the syringe.
4) Transfer the phage “bandate” to a dialysis cassette. Dialyse against 1 litre of
gelatin-free SM buffer containing 1 M NaCl, (add 52 g NaCl per liter buffer) at 4
ºC overnight.
5) Transfer the dialysis cassette to 1 liter of normal gelatin-free SM and dialyze for
2-3 hrs at RT. Repeat.
6) Filter-sterilize the phage prep and store in the dark at 4 ºC.
Center for Phage Technology
Texas A&M University, College Station, TX 77843
Protocol: CsCl phage prep
Rev. 08/18/2011
3 of 3
Download PDF

advertising