Tet-repressible genes - timing

[iddavis at vms.cis.pitt.edu]

In article <4ginp6$asi at panix.com>, iayork at panix.com (Ian A. York) writes:
> 
> Now that it's coming time to test the cells for expression (I 
> plan to test RNA using RT-PCR) I need to know (1) how long the cells need 
> to be tetracycline-free before we can be sure they're on, and (2) 
> whether we need to be anal-retentive about cleaning out the tet.  Do I 
> need to wash out the tet medium, or is it likely to be sufficient to 
> simply remove old medium and replace with new?  

Hi Ian, I've used this system a fair bit.  I didn't formally examine the
questions you raise, but I think it would be wise to be paranoid about
the tet.  It doesn't take a lot to interfere with the assay.  On the other
hand, the whole system can be a little leaky so I'd be interested to 
know just what you are going to do with RT-PCR.  Anyway, I used to
allow at least 24 hours off tet, and usually several days, before testing
(and I was testing for secretion into the supe, so needed a bit longer
to accumulate than some other gene products).  I would wash out the
tet carefully and replace with new.  Remember, though, tet is not stable
in aqueous medium and is light sensitive, so I was probably overzealous.

Hope this is helpful.

Ian Davis				iddavis at vms.cis.pitt.edu