I have been using PEG 8000 (20%) + NaCl to precipitate a clarified phage lysate. After incubating the lysate with the PEG solution, I spin it at 13,000g for 1 hr at 4C. I see the pellets very clearly, but they are hard to resuspend. My instinct is that there is excess PEG in the pellet. It's tricky to resuspend the pellet without shearing the phage. I've seen additional centrifugation and filtering as possible solutions. Does anyone have successful experience with this that can share their solution? Thank you!
First I don't think you need to centrifuge for that long. You may be making a much tighter pellet than you need. I have seen protocols as short as 5 min of centrifugation, but 10-20 is pretty typical. This might also depend on the volume, ie a microfuge on the lower end but a large volume with large bottles might need longer.
The second centrifugation is usually very brief, just to collect the liquid again and remove by pipet. This is to remove any residual PEG solution.
You can just let the pellet sit in buffer for a few hours before trying to resuspend. Depending upon the phage and volume, you can also vortex to resuspend the phage. Again some phage are more tolerant to vortexing than others, but you can quickly test this by seeing if a few minutes of vortexing will reduce PFU of a phage stock.
Do you have any evidence that your resuspension is shearing the phage? Phage are fairly robust in my experience.
I have had good luck with 30 minute spins, aspirating the PPT solution, and letting it sit in buffer (PBST or similar) on ice for 10-30 minutes before resuspending by pipette or moderate vortex.
I have no experience with purification of phage but I've purified a lot of lentivirus over a sucrose cushion. After removing the supernatant we put an excess of PBS into the ultracentrifuge bottles, pack them into wet ice and incubate with gentle shaking in a cold room for several hours, up to overnight, to resuspend the pellet. Then we concentrate the virus by second centrifugation and resuspension in smaller tubes and lower volume.
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