Peg not precipitating out all the phage - (Apr/20/2006 )

I have been having an issue with growing some phages. I add the NaCl and Peg according to Maniatus. After spinning down my Peg - I pour out the supernatant and resuspend my pellet in SM.

Because I have been losing quite a bit of phage during the procedure - I decided to titer at different stages. One of the places I titered was this supernatant after the Peg spin down (which should not contain any phage). It turns out this supernatant contains quite a bit of phage. Why isn't my Peg precitating out all the phage? I have even done experiments where I incubate the Peg at different times (i.e. 2 hours - overnight). There doesn't seem to be much difference.

I have followed the precedure according to Maniatus. Any help would be greatly appreciated.

hi
2 things are sensitive regarding peg. The size of peg 4000 8000 etc...
second one, how old is your peg? PEG tends to loose some properties if too old, as discussed in a thread on this foruum (but i'm not remembering currently where it's )

a point that you may change for :
PEG at 4° or -20° can change some efficiency (except if you're in aqueous solution that freeze )
you may also try glycogen as carrier on overnight precipitation.


Finally, i've done maxipreps that were part done with PEG. mix by inverting the tube 4-5 times is necessary and sufficent.

hope that helps

Thanks so much for the help -

I'm going to try using new PEG (the PEG I used before is at least 2 yrs old).


I'll post again to let everyone interested know how well it worked.