On Thu, 20 Dec 2001, Duncan Clark wrote:
> >In most of the places varying concentration of IPTG has been suggested
> >for induction depending upon the expression system and other things.
> >Most of these are for an low ODs like 0.5-1.
> >
> >In case we need to work on high ODs like 50-100 or 150 would one
You will never get that far for the simple reason that
spectrophotometers peak out at about 3. They will give
values up to about 4, which are meaninglessly beyond
the linear range of the spectrophotometer. OD600 are
absorbance values. Your bugs will already be dying
at about OD600 of about 0.7.
> >require much higher concentration of IPTG and if in what range is
> >generally used for such cultures.
No. This will be useless. There will be so much antibiotic
resistance in the medium that you can clear out nearly 100%
of the antibiotic within minutes! Your soup could quickly
be overrun by bugs spitting out these lethal concentrations
of IPTG. They are quite good at beating this induction
system if given need and opportunity. You give both need
and opportunity with this protocol.
> I've always used the same concentration regardless of OD.
This ends up making sense, but it was not very intuitive to
me at first. I think 100 µM is quite enough for most
systems. I have several different T7 expression bugs. All
of the working ones are killed by only 100 µM IPTG. In fact,
if your bugs still grow well in 1 mM IPTG, your system is
not optimal. Consider a different bug, or subculturing.
> The more critical bit is stage of growth rather than OD itself i.e.
> culture must be in logarithmic growth and not late log or stationary.
I think I would agree, from my own experience. In general, it is
better to have lower OD, below 0.5. Even at 0.5, ampicillin can
be consumed within minutes in a typical T7 system, like BL21(DE3)
and HMS174(DE3) lysogens and proper preparations of these
are pretty much completely killed by 100 to 500 µM IPTG. My
best bugs, are totally killed by this treatment, with no
growth whatsoever if suspensions with this treatment are seeded
onto plates.
Cheers,
Dominic
North 59 37' 30"
East 17 48' 10"
