Help! Panning

Ian A. York york at mbcrr.dfci.harvard.edu
Tue Aug 8 09:56:10 EST 1995


In article <9508080226.AA12954 at gipac.shinshu-u.ac.jp> tnakane at GIPAC.SHINSHU-U.AC.JP (tokio nakane) writes:
>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.

DEAE-dextran does that.  There are two possible ways around it - (1) you
can either tyrypsinize the cells off (trypsin/EDTA, aggressively i.e. 37
oC for 5 - 10 minutes) at 24 hours post-transfection, re-plate the cells,
and then allow them to recover for 2 days before taking them off again.  
(2)  You can use electroporation to transfect them.  This about as 
efficient as DEAE-dextran in our hands, and the cells are much more 
recoverable afterwards.  Also, we found that the dextran altered the cell 
surface markers somewhat in our hands, while the electroporation didn't.  
If you need a protocol for electroporation, please contact me.

Ian

-- 
Ian York   (york at mbcrr.harvard.edu)
Dana-Farber Cancer Institute, 44 Binney St., Boston MA 02115
Phone (617)-632-3921     Fax  (617)-632-2627




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