FRET

[Wolfgang Schechinger]

Dear Vermula, 

are you using pLNCX and pLPCX based vectors (they just come to my mind)? 
Actually, you probably don't need the resistance markers since you easily may 
select for the desired fluorescence (also some sort of resistance marker 
against your scrutinizing eye and/or FACS machine...)

Anyway, they won't harm. And especially if you are using other vectors (means 
no retrovirus) you'll need them in the beginning to kill all non-stable cells.

To select for stable clones, however, you then might seed (in average) single 
cells per well (1 or 2 microplates. Maybe better 1.3 to 1.7 cells per well, 
then the chance to get less empty basins might be higher, I prefer that method 
towards clonong cylinders and so on). Then assay the clones you'll get for 
fluorescence. You may try to grow some clones without antibiotics to see if 
they are really stable. Maybe doesn't work in the case of xFP, since (at least 
in the case of GFP) they have been found to be toxic by some people.

The crucial thing might be how to "titrate" expression levels. In practice,  
you'll get a distribution anyway, dependent on where in their cycle the 
individual cells currently are in. Consulting the lunar calendar might 
appropriate :)

See also some recent postings of mine in this NG (not on lunar calendars). 

Wish you great success, 

Wo

> 
> Hi,
> 
> I am beginning to do some FRET myself. I am planning on doing a double   
> stable cell line which has is resistant to NeoR and PuroR. In other words, one
> plasmid is neoR (contains CFP fusion) and other plasmid is puroR (contains a
> YFP fusion). Is this a bad idea?
> 
> Murali
> 
> 
> ___________________________________________________________________
> Murali Vemula, Ph.D
> Research Fellow
> Harvard Medical School/Shriners Hospital
> 51 Blossom Street
> Boston, MA 02114
> 617-371-4927 (phone)
> 617-371-4951 (fax)
> 
> ---
> 
> 


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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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