Concentrating Lambda Phage
khatipovNO at NOuchicago.edu
Thu Nov 29 18:00:07 EST 2001
It is not very difficult to get 10^10/ml and more from liquid culture. You
might be missing something in the protocol. Important point is to shake
infected cultures pretty fast (~300rpm). Another stage many people neglect
is adding chloroform in the end of lysis and shaking for another 10 min to
release phage from non-lysed cells and debris.
Alternative method that gives high titers (10^10/ml easily) is to use big
15cm plates to propagate the phage. It is a bit more laborious and messy
than liquid culture, but works very reproducibly. Infect cells with enough
phage to produce confluent plaques. Poor top agar with infected cells, grow
O/N to lyse, scrape off top agar, add as little extraction buffer as
possible, treat with chloroform and homogenize it in Dounce homogenizer.
Filter out agar pieces and wash them once to extract entrapped phage.
I am not giving you any protocols here because you can find them in a "Red
book" or elsewhere.
You may try PEG/NaCl precipitation of phage. I did not try that myself, but
it might be worth checking out. PEG precipitation is a standard procedure
used for filamentous phage like M13. However, lambda might be too gentle to
survive in PEG. Actually, I failed to find any reference to using PEG to
concentrate lambda. Please let me know if that worked for you.
As you suggested, centrifugal concentration may also work, but I am not sure
whether there are concentrators for volumes larger than 50 ml. Be careful
though not to contaminate the rotors with phage, because you can imagine
what will happen if someone will be making e.g. competent cells in that
rotor after you.
"neil" <ncsharma at rice.edu> wrote in message
news:3C06684C.F4D8D5F9 at rice.edu...
> I was wondering if anybody has ever tried concentrating lambda
> phage particles to increase titer? Maybe using a spin concentrator or
> similar? Is there a reason to not do this? I am infecting cells with
> CE6 phage (express T7 RNA Polymerase) and would like to boost the the
> current titer (~ 10^9 pfu/mL) to 10^10 or 10^11. The phage are from a
> liquid lysis prep. Thanks!
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