Expression cloning in COS

[Ian A. York]

I'm looking for comments from people who have done expression cloning in 
COS.  We've done a round of selection, and we're not entirely happy with 
what we're getting out; but we don't know what to expect.

We transfected out library in and panned cells 3 days later.  We 
understand that few cells should stick on the first round; however, we 
got quite a few adherent.  Questions:  Should we reduce antibody 
concentration to reduce the background?  How many cells should we expect 
to be seeing?  What kind of false-positive rate is acceptable - or is any 
acceptable?  

We went ahead with Hirt extracts on these cells anyway, mostly for the 
practice.  I didn't get all that many transformed bugs out of it - I 
estimate that the whole extraction would give about 500,000 - 1,000,000 
bacteria (i.e. if I were to transform with every drop).  Is this 
expected?  What kind of numbers should I be expecting here?  (For 
comparison, a pUC control transformation gave about 5 x 10^8 
transformants per ug.)  

I'd appreciate hearing from someone who knows what to expect.  Thanks.

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