Help! Panning

[David J. Evans]

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 
cells.....

I know this won't help detach your cells but it might be a suitable 
alternative approach.

Good luck

David Evans

tnakane at GIPAC.SHINSHU-U.AC.JP (tokio nakane) wrote:
>Hello,
>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.
>
>Tokio    
>




-- 

______________
David J. Evans
AMS, The University of Reading, Whiteknights, P.O. Box 228, READING, UK 
RG6 6AJ
Voice : 44 (0)1734 318893  Fax : 44 (0)1734 316537  
http://www.reading.ac.uk/Virology