SEA-PHAGES | making phage ppt. solution

Link to this post \| posted 05 Nov, 2015 22:49
jross1025	I really need some advice. Tried my hand at making phage precipitating soln last night, got the salt dissolved fine, started to get the PEG into soln. But I can't really get it clarified or even completely dissolved (I think). I'm using the quantities given in the resource guide (19.3 g salt, 30 g PEG 8000, water ultimately to 100ml starting with 60).I tried alternating microwave heating and stirring on a hotplate, then I let it stir without heat for 18 hours or so and it's still opaque. I'm using some that's really old right now–it looks fine, but eventually I'm going to run out of it. It worked when it was new, and I have a hard time believing that anything really happens to this stuff. But again I'm eventually gonna run out one way or another. I have some that's newer but something happened to it in shipment, the bottle wasn't sealed well or something so I've never entirely trusted it. Any advice?

Link to this post | posted 05 Nov, 2015 22:49

I really need some advice. Tried my hand at making phage precipitating soln last night, got the salt dissolved fine, started to get the PEG into soln. But I can't really get it clarified or even completely dissolved (I think). I'm using the quantities given in the resource guide (19.3 g salt, 30 g PEG 8000, water ultimately to 100ml starting with 60).I tried alternating microwave heating and stirring on a hotplate, then I let it stir without heat for 18 hours or so and it's still opaque.

I'm using some that's really old right now–it looks fine, but eventually I'm going to run out of it. It worked when it was new, and I have a hard time believing that anything really happens to this stuff. But again I'm eventually gonna run out one way or another. I have some that's newer but something happened to it in shipment, the bottle wasn't sealed well or something so I've never entirely trusted it.

Any advice?

Link to this post \| posted 06 Nov, 2015 18:49
debbie	Hi Joe, I heard that you spoke with Kevin yesterday about this. I am just adding a link to the protocol that is already on seaphages.org. http://seaphages.org/media/docs/NewDNAProtocol.pdf

Link to this post \| posted 06 Nov, 2015 19:32
jross1025	Thanks Deb (and Kevin), the new protocol looks interesting, however I have a minor question and a major question: Minor question: What is the "precipitate" in the resin(?) that the protocol refers to? I never knew it had any, I mean it has resin beads, but those aren't gonna dissolve in anything, and the bottle is sort of opaque anyway so I'm not quite sure I know what I'm supposed to be seeing dissolving, if that makes any sense. Major question: What about the kids with <5e9 titers? Use the PEG procedure? "Cheat" by centrifugally concentrating (i.e. pellet like you do for EM, and resuspend at lower volume therefore higher titer?)

Link to this post | posted 06 Nov, 2015 19:32

jross1025

Thanks Deb (and Kevin), the new protocol looks interesting, however I have a minor question and a major question:

Minor question: What is the "precipitate" in the resin(?) that the protocol refers to? I never knew it had any, I mean it has resin beads, but those aren't gonna dissolve in anything, and the bottle is sort of opaque anyway so I'm not quite sure I know what I'm supposed to be seeing dissolving, if that makes any sense.

Major question: What about the kids with <5e9 titers? Use the PEG procedure? "Cheat" by centrifugally concentrating (i.e. pellet like you do for EM, and resuspend at lower volume therefore higher titer?)

Link to this post \| posted 06 Nov, 2015 20:04
debbie	Joe, Minor question: The precipitate is the beads. Some folks don't realize that the resin needs to be well(evenly) suspended. Precipitate is probably not the right word. Major question: I would try anything but PEG precipitating. The literature says that you lose about 1/2 of the phage concentration when you do it. So if your lysate is close to 5 x 10^9, then I would just try an extraction. If it is not, concentrate by centrifugation and resuspend in a smaller volume. (and that is not cheating!)

Link to this post | posted 06 Nov, 2015 20:04

debbie

Joe,
Minor question: The precipitate is the beads. Some folks don't realize that the resin needs to be well(evenly) suspended. Precipitate is probably not the right word.
Major question: I would try anything but PEG precipitating. The literature says that you lose about 1/2 of the phage concentration when you do it. So if your lysate is close to 5 x 10^9, then I would just try an extraction. If it is not, concentrate by centrifugation and resuspend in a smaller volume. (and that is not cheating!)

Link to this post \| posted 07 Nov, 2015 02:11
lhughes	Joe: On your original question (in case you end up using the older procedure), I have found that switching the order so that you dissolve the PEG first (slow additions until completely dissolved), then slowly add small amounts of NaCl, that I can get it to dissolve completely without heating. It just takes time. I've never had any luck dissolving the salt first and then the PEG. Lee

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