ethanol treatment vs. plasmid loss

[AKravetz]

In article <39jlnr$ml1 at lastactionhero.rs.itd.umich.edu>, Luis Diaz
<cascas at umich.edu> writes:
>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.



first, a few questions, is this purified plasmid?? and if so, what it
pelleted down by etoh precip or isopropanol precip. I find that you don't
lose that much in 70percent etoh, just watch the time that it is in the
wash. I spin at 15k for 10 minutes then dump out, saving the wash so as
not to lose it if did fall off, good luck and I hope that I am helping...

andy
email at: kravetz at sluvca.slu.edu