(by prae from gmx.net)
Wed Feb 4 05:09:25 EST 2009
dk from no.email.thankstospam.net (DK) wrote:
>In my experience, transformation efficiency of Inoue cells somehow
>deteriorates slowly upon storage at -70C. Unlike electroporation
We usually makes batches, which are used within six month, in this
time period it works. The effiency is probably going down a little
bit, but this was never a problem.
>We do use Inoue protocol in non-critical cases just to save on
>electroporation cuvettes: take electrocompetent cells, mix with
This was the main issue.
>for a little longer at 37C seems to work just as well as 42C. Saves
>from paranoia in the lab about someone "absolutely needing" 42C
>bath NOW :-)
The heat shocking is something religious anyway. Ask two and they will
tell you at least three different times for the heat shock. I found
anything between 30sec and 2min working.
>Can't resist few notes on a protocol:
Thanks for it.
>Gotta love people who swear by OD600. When will
>biologists finally realize that OD600 is partially a function of
It also depends on the photometer.
>Why would any sane biologist filter sterilize pure DMSO?
No. I don't do it either.
>3. Snap freezing. Has anyone ever compared it to just sticking
>cells at -70C? It is well-known that when freezing "real" cells
I haven't compared it for cells, but its worth a try. I have to
convince my colleagues only (can be hard sometimes...).
>cells this way and they work well. I just wonder if the same is
>applicable to Inoue cells.
Probably. Things are often made more complicated than it has to be...
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