David J. Evans
d.j.evans at reading.ac.uk
Thu Aug 10 02:45:18 EST 1995
As an alternative to panning try the CELICS technique we described in :
Ward, T., Pipkin, P.A., Clarkson, N.A., Stone, D.M., Minor, P.D. and
Almond, J.W. Decay accelerating factor (CD55) identified as the receptor
for echovirus 7 using CELICS, a rapid immuno-focal cloning method.
EMBO J. 13, 1994, 5070-5074
It has the advantage over panning that it can be performed in a single
round. It requires the same reagents (plus a micromanipulator) and - for
those readers who aren't going to read the reference above - involves
staining the rare positive cells in a population with b-gal and picking
them out by micromanipulation, extracting the episomal plasmid DNA (see
below) and electroporating it into E.coli.
I should have mentioned that the staining procedure we publish kills the
cells and so episomal vectors must be used. We currently use this
procedure with pCDM8-derived libraries in COS or WOP
I know this won't help detach your cells but it might be a suitable
tnakane at GIPAC.SHINSHU-U.AC.JP (tokio nakane) wrote:
>We are currently trying to clone a G protein-coupled receptor by panning method.
>We constructed a rat brain cDNA library (pME18S vector; SR-alpha promoter)
>and transfected the library to COS-7 cells by DEAE dextran method. After
>72 hrs, we could not detach cells from dishes by the treatment with PBS and
>5 mM EDTA at 37 C for 30 min. The untransfected cells were easily detached
>by the treatment. Any advice would be much appreciated.
David J. Evans
AMS, The University of Reading, Whiteknights, P.O. Box 228, READING, UK
Voice : 44 (0)1734 318893 Fax : 44 (0)1734 316537
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