Normal sterile technique works well, if you practice it a bit more stringently. No standing waste, always bleach the phage immediately after you use it. The stock culture holding the phage/M13KO7 won't have the F plasmid, so the phage physically can't infect them.
On Monday, September 8, 2014 8:16:41 AM UTC-7, Nathan McCorkle wrote:
-- Making sure your stock culture for SS320 is pure is a bit more complex. Making sure it doesn't touch any phage is very important. Use completely different flasks, and make sure to plate negative controls.
If your SS320 stock gets contaminated, beyond control, you just have to get new cells or begin streaking plates for new single colonies. Extensive bleaching and autoclaving can get rid of phage on glassware, but that won't be needed often if you keep your stuff sterile.
I work in a lab that uses this phage, rule of thumb is to always run a negative control when transforming cells (they use SS320 for a different cloning procedure, not this reason). The only real unstable cells are the E coli with the F plasmid, the ones expressing phage should be fine. You *could* retransform it every time, but I've never actually seen anyone do that because the plasmid is 80kb~. Just get new cells
-Koeng
On Monday, September 8, 2014 8:16:41 AM UTC-7, Nathan McCorkle wrote:
On Mon, Sep 8, 2014 at 5:24 AM, Koeng <koen...@gmail.com> wrote:
> The phage only get synthesized in the presence of M13KO7 and only infect in
> the presence of the F plasmid. The shipped phage won't have either one.
>
> However, you'd have to make sure your stock of F plasmid E coli (SS320)
> don't get contaminated.
So do you transform with F plasmid prior to phage use every time to
prevent contamination of your untransformed stock culture?
Have you come upon any means of curing the culture of the phage? The
rifamycin seems to only stop synthesis, but the paper didn't mention
anything about catabolism/disintegration of the phage particles.
If you don't do this, do you have a lab room that is devoted to phage
work? And have a different room for your stock culture operations?
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