Electroporation

[Christian Praetorius]

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 
>"competent" cells. 

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...

Christian

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