ethanol treatment vs. plasmid loss

[Shahram Mori]

Luis Diaz (cascas at umich.edu) wrote:
: I have an mAb that recognizes an intracellular epitope of a T cell
: membrane glycoprotein.  I want to clone the protein this mAb recognizes
: by transfecting a T cell cDNA library into NIH 3T3 cells and screening by
: flow cytommetry (FACS) with our FITC-labeled mAb.  The problem I am
: concerned about is that to screen with my mAb we have to first
: permeabilize the transfected cells with 70% ethanol.  I am worried that a
: successfully transfected plasmid would be lost after ethanol treatment. 
: Am I wrong to assume the loss of a plasmid after 70% ethanol treatment or
: is this something that is likely to occur.

: Luis A. Diaz
: Medical Student 
: University of Michigan
I would think that Permealizing doesn't mean total lysis of the cell. If 70%
ethanol doesn't lyse it, I would guess it should be alright. 
Cheers,
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003