View source for PhiX:Stock preparation

==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'''<br>
People use various tools to lever a plaque out from a plate (e.g., you can bend an inoculation loop through 90<sup>o</sup>. 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.<br>
Before starting the following are required:<br>
1. For picking: sterile Pasteur pipettes (one per plaque), plenty of sterile toothpicks (autoclave in small beaker), sterile razor blades and cutting surface.<br>
2. Then: labelled sterile 1.5ml microtubes (one per plaque) containing 1100ul HFB or LB buffer.<br>
3. For purifying: supply of chloroform, labelled sterile 1.5ml microtubes (one per plaque) containing 70ul of DMSO.<br>
4. Keep all these components and tubes accessible. Work near a fume hood (required for purifying the plaque).

'''Picking a Plaque'''<br>
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).<br>
6. Apply and hold a slight squeeze in the bulb of the pipette and lower (at 90<sup>o</sup>) down onto agar surface piercing the top agar layer.<br>
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!<br>
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.<br>

'''Purifying the Plaque'''<br>
9. In a fume hood add 110ul (10%v/v) chloroform (you could use less) and close lid (note chloroform has low viscosity).<br>
10. Vortex the tube to mix the phases, kill bacteria and liberate the phage.<br>
11. Centrifuge at full speed (16,000 rcf) for 4 minutes.<br>
12. Remove the tube from the centrifuge gingerly and check that the phases are separated.<br>
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.<br>
14. Freeze at -80<sup>o</sup>C (we have not tested -20<sup>o</sup>C extensively for long-term storage, but it is better than nothing).

==Amplifying Phage==
'''Preparing a lot of Phage'''<br>
Some cultures will not clear, but will still contain phage. A second infection can be initiated...<br>
1. Prepare plenty of LB with 2mM CaCl<sub>2</sub> and 10mM MgCl<sub>2</sub>.<br>
2. Inoculate 15ml of LB with a colony of <i>E. coli</i>; use a baffled conical flask.<br>
(2b. Quick option: add 3ml of overnight culture to 12ml LB; use a 50ml centrifuge tube.)<br>
3. Grow to an O.D.<sub>600nm</sub> of ~0.7 (~3 hours for quick option).<br>
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.<br>
5. Leave culture for many hours (e.g., overnight). The culture is not likely to clear, but you can follow O.D.<sub>600nm</sub> if you wish (against a control culture).<br>

'''Purifying the Lysate'''<br>
This part is quite scaleable.<br>
6. Aliquot a decent volume of lysate (e.g., 1.1ml) into a suitable sterile container (e.g., a micro tube).<br>
7. In a fume hood, add 10% v/v chloroform (e.g., 110ul).<br>
8. Vortex the tube to mix the phases and lyse residual bacterial hosts.<br>
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).<br>
10. Remove the tube from the centrifuge gingerly and check that the phases are separated.<br>
11. Aliquot most (e.g., 930ul) of the aqueous phase (top layer) into a new container.<br>
The next steps depend on your application...