Editing Phage Purification (section)

== Troubleshooting ==

'''Low virion counts'''

If your phage yields are too low, e.g. less than 0.1 O.D.x mL in the original culture, most likely virions are embedded with bacterial debris, the U.V. spectrum will exhibit a shallower minimum or worst, no minimum below 269 nm, and the concentration of virions measured by this method will be erroneous. It is technically difficult to purify phage further by PEG-precipitation in this situation.

To improve phage yields and take full advantage of this technique, 1) if you are using a helper phage, make sure that the density of the bacterial culture is optimal at the time of superinfection (e.g. around 0.4-0.5 OD600 for TG1 and SS320), 2) if you are using M13KO7 as a helper phage, switch to a more efficient packager such as CM13, and 3) more importantly, try to switch to a phage display system like the pADL Phagemid Vector System where expression is under tight control. Many old phagemid systems such as pComb3 or Phen2 vectors use a very leaky lac promoter driving overexpression of the fusion protein even in absence of IPTG, resulting in bacterial toxicity, slow bacterial growth, bacterial lysis and limited phage production.

'''Centrifugation Table'''

The protocol can be easily adapted to larger scale preparations by adjusting the centrifugation time while keeping the relative centrifugal force to 13,000 g as indicated in the following table:

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Preparation&nbsp;<br/>
| mini (1-2 ml)&nbsp;<br/>
| midi (20-50 ml)&nbsp;<br/>
| maxi (100-400 ml)<br/>
|-
| Bacterial Clearance<br/>
| 2 min&nbsp;&nbsp;<br/>
| 3 min&nbsp;<br/>
| 1 min&nbsp;<br/>
|-
| Centrifugation<br/>
| 5 min<br/>
| 10 min&nbsp;<br/>
| 5 min&nbsp;<br/>
|-
| Clearing&nbsp;<br/>
| 10 min&nbsp;&nbsp;<br/>
| 20 min&nbsp;<br/>
| 10 min<br/>
|}


[[Category:Antibodies]][[Category:Protocols]]