Difference between revisions of "PhiX:Stock preparation"

Revision as of 05:22, 23 February 2017

Summary

A method is required to prepare a reasonably high titer stock of virus. This method is a rough and ready. PEG-based methods are superior for purification. You may start with a plaque for this or with a small volume of less concentrated stock.

Plaque Harvesting

Requirements
People use various tools to lever a plaque out from a plate (e.g., you can bend an inoculation loop through 90^o. For this method a supply of Pasteur pipettes, razor blades and toothpicks are required. Some folk also 25% glycerol as a cryoprotectant; if you do this, use a 1ml syringe because it is highly viscous and difficult to pipette.
Before starting the following are required:
1. For picking: sterile Pasteur pipettes (one per plaque), plenty of sterile toothpicks (autoclave in small beaker), sterile razor blades and cutting surface.
2. Then: labelled sterile 1.5ml microtubes (one per plaque) containing 1100ul HFB or LB buffer.
3. For purifying: supply of chloroform, labelled sterile 1.5ml microtubes (one per plaque) containing 70ul of DMSO.
4. Keep all these components and tubes accessible. Work near a fume hood (required for purifying the plaque).

Picking a Plaque
5. Taking care to stay sterile, cut the end off of a Pasteur pipette (usually to second gradation from the tip - but try to match bore size after sectioning to diameter of plaques).
6. Apply and hold a slight squeeze in the bulb of the pipette and lower (at 90^o) down onto agar surface piercing the top agar layer.
7. Slowly release the tension a little (not all the way) and pull the pipette upwards. Pay attention to the effect - you want to carry the plug of agar up without launching it up into the bulb of your pipette!
8. Move over to a tube containing 1100ul LB/HFB and apply pressure again to the bulb to release the plug of agar into the medium.

Purifying the Plaque
9. In a fume hood add 110ul (10%v/v) chloroform (you could use less) and close lid (note chloroform has low viscosity).
10. Vortex the tube to mix the phases, kill bacteria and liberate the phage.
11. Centrifuge at full speed (16,000 rcf) for 4 minutes.
12. Remove the tube from the centrifuge gingerly and check that the phases are separated.
13. Aliquot 930ul (most) of the aqueous (top) phase into the second microtube containing DMSO (=cryoprotectant). Take care not to disturb the interphase layer when you do this.
14. Freeze at -80^oC (we have not tested -20^oC extensively for long-term storage, but it is better than nothing).

Amplifying Phage

Preparing a lot of Phage
Some cultures will not clear, but will still contain phage. A second infection can be initiated...
1. Prepare plenty of LB with 2mM CaCl₂ and 10mM MgCl₂.
2. Inoculate 15ml of LB with a colony of E. coli; use a baffled conical flask.
(2b. Quick option: add 3ml of overnight culture to 12ml LB; use a 50ml centrifuge tube.)
3. Grow to an O.D._600nm of ~0.7 (~3 hours for quick option).
4. Inoculate culture with phage from frozen or fresh stocks. The more phage added the better (e.g., ~1ml), depending on how much you've got.
5. Leave culture for many hours (e.g., overnight). The culture is not likely to clear, but you can follow O.D._600nm if you wish (against a control culture).

Purifying the Lysate
This part is quite scaleable.
6. Aliquot a decent volume of lysate (e.g., 1.1ml) into a suitable sterile container (e.g., a micro tube).
7. In a fume hood, add 10% v/v chloroform (e.g., 110ul).
8. Vortex the tube to mix the phases and lyse residual bacterial hosts.
9. Centrifuge at full speed (16,000 rcf) for 4 minutes (if you are processing a larger volume, a slower but longer spin in a larger centrifuge may be required).
10. Remove the tube from the centrifuge gingerly and check that the phases are separated.
11. Aliquot most (e.g., 930ul) of the aqueous phase (top layer) into a new container.
The next steps depend on your application...